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Laboratory A1C does not precisely reflect glycemic control in those with altered RBC lifespan and our aim was to evaluate an adjusted A1C, a glycemic measure that adjusts A1C to a reference RBC lifespan. By removing the individual RBC lifespan variation, adjusted A1C better reflects intracellular glucose exposure in RBCs and perhaps other organs susceptible to complications. Iron-deficiency anemia and treatment is known to affect RBC lifespan and therefore provides opportunity to evaluate the model. The patient was a 53-year-old woman with iron-deficiency anemia and type 1 diabetes utilizing sensor-augmented pump therapy. Longitudinal A1C and CGM were evaluated with a novel kinetic model to estimate RBC lifespan and glucose uptake rate. During the six months before iron therapy, the RBC lifespan was estimated at 151 days and 93% of a reference glucose uptake. During six months of iron therapy, the RBC lifespan decreased to 122 days, while the RBC glucose uptake increased slightly to 101% of reference value. The RBC lifespan change explains the observed laboratory A1C change (Figure) and can be attribute to the iron therapy and supported by Hb, RDW and Ferritin data. The adjusted A1C based on these two different RBC lifespans agree with the stable CGM mean glucose of this patient. In this case study, we confirmed the clinical relevance of adjusted A1C to account for variations in RBC lifespan. Disclosure Y.Hirota: Speaker's Bureau; Abbott Japan Co., Ltd., Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Daiichi Sankyo, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Kirin Co., Ltd., Lilly, Medtronic, Mochida Pharmaceutical Co., Ltd., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Terumo Corporation. Y.Xu: Employee; Abbott Diabetes. A.Yamamoto: None. A.Matsuoka: None. T.Dunn: Employee; Abbott. W.Ogawa: Advisory Panel; Abbott Japan Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk, Other Relationship; Abbott Japan Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Nippon Boehringer Ingelheim Co. Ltd., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Research Support; Abbott, Astellas Pharma Inc., Eli Lilly and Company, Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Funding Abbott Diabetes Care

Objective: In addition to the inhibition of gluconeogenesis in the liver, metformin exerts various effects on the gut that likely contribute to the lowering of blood glucose levels. Positron emission tomography (PET)-computed tomography recently has revealed that metformin promotes the intestinal accumulation of [18F]fluorodeoxyglucose (FDG), a nonmetabolizable glucose derivative, indicating that the drug affects glucose handling in the intestine. It has remained unknown, however, whether this accumulation occurs in the wall or intraluminal space of the intestine. We here addressed this question with the use of [18F]FDG-based PET-magnetic resonance imaging (MRI), a recently developed imaging modality with increased accuracy of registration and high soft-tissue contrast. Methods: Among 244 individuals with type 2 diabetes who underwent PET-MRI, we extracted 24 pairs of subjects matched for age, BMI, and HbA1c level who were (metformin group) or were not (control group) receiving treatment with metformin. We evaluated accumulation of [18F]FDG in different portions of the intestine with both a visual scale and measurement of maximum standardized uptake value (SUVmax), and such accumulation within the intestinal wall or lumen was discriminated on the basis of SUVmax. Results: SUVmax of the jejunum, ileum, and right or left hemicolon was greater in the metformin group than in the control group. [18F]FDG accumulation in the ileum and right or left hemicolon as assessed with the visual scale was also greater in the metformin group. SUVmax for the intraluminal space of the ileum and right or left hemicolon, but not that for the intestinal wall, was greater in the metformin group than in the control group. Conclusions: Metformin treatment was associated with increased accumulation of [18F]FDG in the intraluminal space of the intestine, suggesting that this drug promotes the transport of glucose from the circulation into stool. Disclosure K. Sakaguchi: Other Relationship; Self; AstraZeneca, Novartis AG, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Y. Morita: None. M. Nogami: None. Y. Okada: None. Y. Hirota: Other Relationship; Self; Sanofi. K. Sugawara: None. Y. Tamori: None. T. Murakami: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Background: Insulin degludec (IDeg) and insulin glargine U300 (IGla-300) are recently launched ultra-long-acting insulin formulations. It remains unclear whether or how their glucose-stabilizing effects differ. We now compared the effects of these insulins on parameters on glycemic variability in patients with type 1 diabetes (T1D). Methods: This multicenter, randomized, crossover, non-inferiority trial recruited 46 patients with T1D treated with basal-bolus insulin therapy at 14 facilities in Japan. Patients were randomly assigned 1:1 to IGla-300 (first period)/IDeg (second period) or IDeg (first period)/IGla-300 (second period) groups, in which subjects were treated with the corresponding basal insulin for 4-week periods. The last week of each treatment period constituted the data collection phase, during which the participants determined the plasma glucose level seven times a day using a SMBG devise and were also equipped with a CGM devise. The primary end point was comparison of day-to-day glycemic variability evaluated by the standard deviation (SD) of FPG during the last week of each treatment period. Results: The SD for FPG in the IDeg treatment period was not inferior to that in the IGla-300 treatment period (95% CI, -16.1-3.0 mg/dL). The levels (95% CI, -18.4-5.7 mg/dL) and the coefficient of variance (95% CI, -4.6-4.3 %) of FPG did not significantly differ between the two periods. Parameters obtained with CGM, including time-in-range, duration of nocturnal hypoglycemia, mean amplitude of glycemic excursion, and mean of the daily differences, as well as the doses of basal and bolus insulins also did not significantly differ between the two treatment periods. Conclusion: IDeg was not inferior to IGla-300 in terms of inter-day glycemic variability in the treatment of T1D, and the treatments with these two insulins yielded similar outcomes for various parameters related to blood glucose control. Disclosure H. Miura: None. K. Sakaguchi: Other Relationship; Self; AstraZeneca, Novartis AG, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. A. Kaneko: None. J. Ito: None. Y. Morita: None. T. Yamada: None. N. Otowa-Suematsu: None. A. So: None. T. Nakamura: None. H. Komada: None. Y. Okada: None. Y. Hirota: Other Relationship; Self; Sanofi. Y. Tamori: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Background: A protocol of differentiation form induced pluripotent stem cells (iPSCs) to hepatocytes has been previously reported, and such iPSCs-derived hepatocytes possessed morphological and biochemical characteristics similar to authentic hepatocytes. It is however unknown whether iPSCs-derived hepatocytes normally respond to metabolic regulatory hormones including insulin. Mutations in the gene for p85α subunit of PI3K (PIK3R1), an essential element of insulin action, give rise to severe insulin resistance in human. Such cases are very rare, and only ∼30 families with PIK3R1 mutations have been reported as of today. Methods and Results: We established a protocol of differentiation from iPSCs to hepatocytes that respond to insulin. In hepatocytes differentiated from iPSCs derived from healthy subject with this protocol, phosphorylation of AKT and the expression of the lipogenic genes FASN as well as SREBF1c were induced by insulin. Furthermore, the expression of the gluconeogenic gene G6PC was upregulated by a combination of cAMP and dexamethasone, which was markedly impaired by the addition of insulin. We then generated iPSCs from a severe insulin resistant diabetic patient with the PIK3R1 c.1945C>T mutation, a hot-spot mutation in this gene. Insulin-induced gene expression along with phosphorylation of AKT was impaired in hepatocytes differentiated from the patient-derived iPSCs. We also repaired the mutation of PIK3R1 in the patient-derived iPSCs by the CRISPR/Cas9 genome editing. Insulin responsiveness was recovered in hepatocytes differentiated from the resultant iPSCs in which the mutation was repaired. Conclusion: We established a differentiation protocol of hepatocytes in which insulin’s metabolic actions can be evaluated. The usefulness of this protocol was validated with the use of iPSCs derived from a severe insulin-resistant patient with PIK3R1 mutation. Disclosure T. Hamaguchi: None. Y. Hirota: Other Relationship; Self; Sanofi. T. Takeuchi: None. M. Koyanagi-Aoi: None. T. Aoi: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Aims: Urinary glucose excretion (UGE) induced by SGLT2 inhibitors (SGLT2i) varies considerably by individual, and is influenced by blood glucose level and glomerular filtration rate (GFR). It is however unknown what other factors are involved in this process. We have now investigated the relation between various physiological parameters and UGE in patients treated with an SGLT2i. Methods: To minimize the influence of glycemic variability, we analyzed SGLT2i-induced UGE during a hyperinsulinemic-euglycemic clamp. OGTT and a 120 min-hyperinsulinemic-euglycemic clamp, during which plasma glucose (PG) were maintained at 90 mg/dl, were performed with type 2 diabetes subjects (n=45) within 6 days after the start of dapagliflozin (5mg/day). Relationships between UGE during the clamp and various indexes for insulin secretion/sensitivity were analyzed. Disposition index (DI) was calculated as the product of insulinogenic index (II) and insulin sensitivity index (ISI) determined by the clamp. Results: During the clamp, UGE considerably varied from 28.2 to 176.9 (mean ± SD, 87.1 ± 32.8) mg/kg/120min. In univariate analysis, UGE was not correlated with PG before the clamp, mean PG during the clamp or any indexes for insulin secretion/sensitivity determined by OGTT or the clamp (HOMA-β, II, OGTT AUCIRI/AUCPG, HOMA-IR, composite index, QUICKI and ISI). However, UGE was correlated with log-transformed DI (r = -0.338, p = 0.023) as was eGFR (r = 0.418, p = 0.004). Multivariate analysis revealed that eGFR and log-transformed DI were independent predictors of UGE (R2 = 0.267, β = 0.332, p = 0.045, and β = -0.315, p = 0.031, respectively). Conclusion: Even in euglycemic conditions, SGLT2i-induced UGE considerably varied by individuals and was correlated with DI, which represents individual capacity of glucose disposal. The present results thus suggest that SGLT2i-induced UGE is regulated by an unidentified physiological process that is related to glucose tolerance in living body. Disclosure K. Sakaguchi: Other Relationship; Self; AstraZeneca, Novartis AG, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. A. So: None. J. Ito: None. A. Kaneko: None. Y. Morita: None. T. Yamada: None. H. Miura: None. N. Otowa-Suematsu: None. T. Nakamura: None. H. Komada: None. Y. Okada: None. Y. Hirota: Other Relationship; Self; Sanofi. Y. Tamori: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Despite recent tightening of cutoffs for hypertension (HT) in major global guidelines, guidelines, especially according to GTS, remain debatable. We analyzed data from a nationwide claims database on 293,396 participants without prior CAD or CVD from 2008-16. Multivariate Cox regression model identified risks of CAD and CVD events. Hazard ratios (HRs) were compared among 5 levels of systolic blood pressure (SBP) (≤119, 120-129, 130-139, 140-149, ≥150) according to GTS [i.e., normal glucose tolerance (NGT), prediabetes (pre-DM), diabetes (DM)]. During the study period, 1099 CAD and 1990 CVD events occurred. Those with SBP levels ≥120 mmHg had a significantly higher risk of incident CAD/CVD than those with lower levels. The incidence of CAD and CVD did not differ significantly between NGT and pre-DM in those with SBP ≤119 whereas HRs for incident CAD for DM was almost 2.7, which was similar to NGT and pre-DM with SBP ≥150 mmHg (Table). Compared with NGT and SBP ≤119 mmHg, the HR for incident CAD among DM and SBP ≥150 mmHg was 5.91 (4.22-8.26), while SBP ≥150 mmHg significantly increased the risk of CVD about 4 fold regardless of GTS. These results based on a large database implied the need for even lower BP targets regardless of GTS. The impact of BP was stronger in DM in preventing CAD. Disclosure M.H. Yamada: None. K. Fujihara: None. Y. Yaguchi: None. T. Osawa: None. M. Kitazawa: None. Y. Matsubayashi: None. M. Iwanaga: None. T. Yamada: None. N. Yamanaka: None. H. Seida: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. H. Sone: Research Support; Self; Kyowa Hakko Kirin Co., Ltd., Novartis AG, Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co. Funding 18K17897

Although the 2017 ACC/AHA guidelines indicated a generalized BP goal of less than 130/80 mm Hg, the impact of BP separately on CAD or CVD in people with different GTSs has yet to be clarified. To clarify this, data from a nationwide claims database involving 293,396 participants without prior CAD or CVD during 2008-16 were analyzed. Multivariate Cox regression model was used to identify risks of a CAD and/or CVD event. Hazard ratios (HRs) were compared among 4 groups of participants divided according to stratified levels of BP [i.e., 90 mmHg, ≥140/90 mmHg] according to GTS [i.e., normal glucose tolerance (NGT), prediabetes (pre-DM), diabetes mellitus (DM)]. During the study period, 1099 CAD and 1990 CVD events occurred. Despite that a significant trend between BP and incident CAD was observed regardless of GTS, risk for CVD was dependent on BP only in the NGT and pre-DM groups. Moreover, there were no significant differences in CVD risks between categories of BP Disclosure M. Harada: None. K. Fujihara: None. M. Yamamoto: None. M. Kaneko: None. M. Kitazawa: None. H. Suzuki: None. Y. Matsubayashi: None. T. Yamada: None. N. Yamanaka: None. H. Seida: None. W. Ogawa: Consultant; Self; Abbott. Research Support; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Daiichi Sankyo Company, Limited, Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Lilly Diabetes, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd. H. Sone: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited.

Although GTS, prior CAD and other traditional risk factors independently affect incidence of subsequent CAD, their impact in the same cohort has been scantly studied. We analyzed a nationwide claims-based database of 138,162 men aged 18-72 y in 2008-14. Participants were classified as normal glucose tolerance (NGT, n=78,230), prediabetes (PreDM, n=45,610) or diabetes mellitus (DM, n=14,322) based on blood tests and prescriptions and with prior (CAD+) or without prior CAD (CAD-) based on ICD-10 codes and questionnaires. Among CAD-, management of traditional risks differed from those with and without CAD events, while such differences were weaker in preDM and DM with CAD+. Cox regression model showed that PreDM and DM confer approximately 1.2- and 2.8-fold excess risk of CAD, respectively, compared with CVD- with NGT. CAD+ confers approximately a 5- to 8-fold increased risk. Most traditional risks were independently associated with subsequent CAD in the CAD- group regardless of GTS. Conversely, in the CAD+ group, their impact was weakened and only HDL-C was a significant risk factor for future CAD in those with DM and CAD+ (Table). Thus, the impact of known risks on subsequent CAD differs markedly according to GTS and CAD+. Those with CAD+ and glucose abnormality, especially DM, need treatment residual risk factors as well as traditional risk factors. Disclosure M. Kitazawa: None. K. Fujihara: None. T. Osawa: None. M. Yamamoto: None. M. Harada: None. M. Kaneko: None. Y. Matsubayashi: None. T. Yamada: None. N. Yamanaka: None. H. Seida: None. W. Ogawa: Consultant; Self; Abbott. Research Support; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Daiichi Sankyo Company, Limited, Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Lilly Diabetes, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd. H. Sone: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited.

Although adipocytes are major targets of insulin, the influence of impaired insulin action in adipocytes on metabolic homeostasis has been unclear. To provide insight into the role of insulin action in adipocytes, we generated mice that lack 3'-phosphoinositide-dependent kinase1 (PDK1) specifically in adipocytes (A-PDK1KO mice). These mice manifest impairment of metabolic actions of insulin in adipose tissue and a reduction in adipose tissue mass. The mice developed insulin resistance, glucose intolerance, and hepatic steatosis, all of which were markedly attenuated by additional ablation of FoxO1 specifically in adipocytes without an effect on adipose tissue mass, indicating that the PDK1-FoxO1 axis in adipocytes plays an important role in systemic metabolic homeostasis through a mechanism independent of the regulation of adipose tissue mass. Neither circulating levels of adiponectin and leptin nor inflammation in adipose tissue differed between A-PDK1KO mice and adipocyte-specific PDK1/FoxO1 deficient mice (A-PDK1/FoxO1KO mice), indicating that those factors did not account for the difference in metabolic phenotypes of the two genotypes of mice. Microarray and lipidomics analyses revealed that leukotriene B4 (LTB4) levels in plasma and in adipose tissues as well as the expression of 5-lipoxygenase (5-LO), a rate limiting enzyme for LTB4 production, in adipose tissues were increased and normalized in A-PDK1KO and A-PDK1/FoxO1KO mice, respectively. Administration of a 5-LO inhibitor reduced plasma levels of LTB4 and ameliorated insulin resistance and glucose intolerance in A-PDK1KO mice. Furthermore, insulin was found to inhibit LTB4 production through downregulation of 5-LO expression via the PDK1-FoxO1 pathway in isolated adipocytes. These results thus indicate that insulin signaling via the PDK1-FoxO1 pathway negatively regulates the production of LTB4 through the suppression of 5-LO in adipocytes and thereby maintains systemic insulin sensitivity. Disclosure T. Hosooka: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. W. Ogawa: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Abbott.

Background: Evidence suggests that diabetes is a promoting factor of sarcopenia. Mechanism how the condition accelerates the development of sarcopenia remains ambiguous. Results: In streptozotocin (STZ)-induced diabetic mice, skeletal muscle mass was decreased by ∼15 % within 21 days after the STZ treatment. The protein abundance of transcription factor KLF15 as well as the mRNA abundance of proteins related to muscle atrophy (Foxo3a, Atrogin1 and Murf1) were elevated whereas the mRNA of KLF15 was unaltered in skeletal muscle of STZ-diabetic mice. Decrease in skeletal muscle mass and increase in the mRNA abundance of muscle atrophy-related proteins triggered by STZ-induced diabetes were prevented in muscle-specific KLF15 deficient mice suggesting that KLF15 plays a key role in diabetes-induced muscle atrophy. Treatment of C2C12 cells with high concentration of glucose (25 mmol/l) decreased the ubiquitination of and increased the protein abundance of KLF15. The E3 ubiquitin ligase WWP1 was found to be downregulated in skeletal muscle of STZ- diabetic mice and in C2C12 cells treated with glucose. Overexpression and knockdown of WWP1 in C2C12 cells resulted in the decrease and the increase, respectively, of KLF15 protein without affecting its mRNA expression. The mRNA abundance of WWP1 in skeletal muscle was downregulated and the mass of skeletal muscle was reduced in Akita mice, which develop diabetes due to beta-cell dysfunction. Administration of the SGLT2 inhibitor empagliflozin, which lowers glycemia without affecting insulin levels, to Akita mice upregulated the expression of WWP1 in skeletal muscle and increased the skeletal muscle mass of these mice. Conclusion: Our results suggest that hyperglycemia upregulates the protein abundance of KLF15 via the downregulation of WWP1, which catalyzes the ubiquitination of KLF15. Hyperglycemia-induced upregulation of KLF15 leads to the induction of genes for muscle atrophy-related proteins, which in turn promotes the atrophy of skeletal muscle. Disclosure Y. Hirata: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. K. Nomura: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Y. Senga: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. M. Imamura: None. S. Takeda: None. Y. Okada: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. S.J. Burden: None. T. Hosooka: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. W. Ogawa: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Abbott.

Interactions of pancreatic islets and islet-associated mononuclear cells (IAMCs) from the nonobese diabetic (NOD) mouse were morphologically investigated. To obtain IAMCs, pancreatic islets isolated from adult NOD mice were cultured for 7 days with interleukin 2. Noted by light microscopy, interactions between IAMCs and freshly isolated islets from young NOD mice began 30 min after the initiation of the coculture, and 6 h later, normal cellular array of the islets was lost. By electron microscopy, most IAMCs had low nucleus-cytoplasm ratio, the nucleus was notched and exhibited condensed chromatin along the nuclear membrane, and well-developed Golgi complexes and several mitochondria were distributed in the cytoplasm. These IAMCs adhered to beta-cells, but not to alpha- or delta-cells, with their pseudopods and caused cytolysis of beta-cells. Immunohistochemical study with antibodies specific for pancreatic hormones demonstrated that only cells reacting with anti-insulin antibody were selectively lost as the incubation time proceeded. Electron immunohistochemistry by immunogold technique showed that effector cells in IAMCs reacted with anti-CD8 (Lyt-2) antibody, but not anti-CD4 (L3T4) or anti-asialogangliosideM1 antibody. In addition, the concentration of pancreatic hormones in the culture medium, used as a marker of cytolysis, also demonstrated that insulin was significantly increased after 6 h of culture, whereas glucagon and somatostatin were not. These results suggest that CD8+ cytotoxic T lymphocytes are involved in the selective destruction of pancreatic beta-cells in the NOD mouse.

The immune mechanisms directly responsible for β-cell destruction in insulin-dependent diabetes are undefined. We studied the role of MHC class I–restricted T lymphocytes in the development of diabetes in cyclophosphamide (CY)-treated male and untreated female NOD mice (H-2Kd,Db). After administration of CY to 10-wk-old male NOD/Shi/Kbe mice, 37 of 64 (58%) phosphate-buffered saline–injected control mice and 13 of 22 (59%) anti-Kb and 12 of 27 (44%) anti-Db monoclonal antibody (MoAb)-injected mice became diabetic by 14 wk of age, whereas only 3 of 38 (8%) anti-Kd and 2 of 13 (15%) anti-Lyt-2 MoAb-injected mice did. In untreated female NOD/Shi/Kbe mice, 30 of 46 (65%) mice developed spontaneous diabetes by 30 wk of age, whereas none of 9 anti-Kd MoAb-injected mice became diabetic. Immunohistochemical studies showed that islet-infiltrating cells in CY-treated control mice were composed mainly of both L3T4+ and Lyt-2+ T lymphocytes, whereas many L3T4+ and very few Lyt-2+ lymphocytes infiltrated within the islets in anti-Kd MoAb-injected mice. Administration of anti-Lyt-2 MoAb induced the absence of Lyt-2+ T lymphocytes in the islet and spleen. However, anti-Kd MoAb did not change the number of spleen cells or the T-lymphocyte subset and response to concanavalin A. These results suggest that MHC class I Kd-restricted Lyt-2+ T lymphocytes play an important role as direct effector cells in destruction of β-cells in NOD/Shi/Kbe mice.

Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase–dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes–like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.

The mechanism by which insulin induces the expression of the sterol regulatory element binding protein 1c (SREBP-1c) and glucokinase genes was investigated in cultured rat hepatocytes. Overexpression of an NH2-terminal fragment of IRS-1 that contains the pleckstrin homology and phosphotyrosine binding domains (insulin receptor substrate-1 NH2-terminal fragment [IRS-1N]) inhibited insulin-induced tyrosine phosphorylation of IRS-1 as well as the association of IRS-1 with phosphatidylinositol (PI) 3-kinase activity, whereas the tyrosine phosphorylation of IRS-2 and its association with PI 3-kinase activity were slightly enhanced. The equivalent fragment of IRS-2 (IRS-2N) prevented insulin-induced tyrosine phosphorylation of both IRS-1 and IRS-2, although that of IRS-1 was inhibited more efficiently. The insulin-induced increases in the abundance of SREBP-1c and glucokinase mRNAs, both of which were sensitive to a dominant-negative mutant of PI 3-kinase, were blocked in cells in which the insulin-induced tyrosine phosphorylation of IRS-1 was inhibited by IRS-1N or IRS-2N. A dominant-negative mutant of Akt enhanced insulin-induced tyrosine phosphorylation of IRS-1 (but not that of IRS-2) and its association with PI 3-kinase activity, suggesting that Akt contributes to negative feedback regulation of IRS-1. The Akt mutant also promoted the effects of insulin on the accumulation of SREBP-1c and glucokinase mRNAs. These results suggest that the IRS-1–PI 3-kinase pathway is essential for insulin-induced expression of SREBP-1c and glucokinase genes.