Akita Mice Research Articles

TNF, but not hyperinsulinemia or hyperglycemia, is a key driver of obesity-induced monocytosis revealing that inflammatory monocytes correlate with insulin in obese male mice.

Inflammation contributes to obesity‐related hyperinsulinemia and insulin resistance, which often precede type 2 diabetes. Inflammation is one way that obesity can promote insulin resistance. It is not clear if the extent of obesity, hyperinsulinemia, or hyperglycemia, underpins changes in cellular immunity during diet‐induced obesity. In particular, the requirement for obesity or directionality in the relationship between insulin resistance and monocyte characteristics is poorly defined. Inflammatory cytokines such as tumor necrosis factor (TNF) can contribute to insulin resistance. It is unclear if TNF alters monocytosis or specific markers of cellular immunity in the context of obesity. We measured bone marrow and blood monocyte characteristics in WT and TNF −/− mice that were fed obesogenic, high fat (HF) diets. We also used hyperglycemic Akita mice and mice implanted with insulin pellets in order to determine if glucose or insulin were sufficient to alter monocyte characteristics. We found that diet‐induced obesity in male mice increased the total number of monocytes in blood, but not in bone marrow. Immature, inflammatory (Ly6Chigh) monocytes decreased within the bone marrow and increased within peripheral blood of HF‐fed mice. We found that neither hyperinsulinemia nor hyperglycemia was sufficient to induce the observed changes in circulating monocytes in the absence of diet‐induced obesity. In obese HF‐fed mice, antibiotic treatment lowered insulin and insulin resistance, but did not alter circulating monocyte characteristics. Fewer Ly6Chigh monocytes were present within the blood of HF‐fed TNF −/− mice in comparison to HF‐fed wild‐type (WT) mice. The prevalence of immature Ly6Chigh monocytes in the blood correlated with serum insulin and insulin resistance irrespective of the magnitude of adipocyte or adipose tissue hypertrophy in obese mice. These data suggest that diet‐induced obesity instigates a TNF‐dependent increase in circulating inflammatory monocytes, which predicts increased blood insulin and insulin resistance independently from markers of adiposity or adipose tissue expansion.

Pulmonary Vein Ganglia Are Remodeled in the Diabetic Heart.

BackgroundCardiac autonomic neuropathy is thought to cause adverse cardiovascular effects in diabetes mellitus. Pulmonary vein ganglia (PVG), which have been implicated in normal and abnormal heart rhythm regulation, have not been fully investigated in type 1 diabetes mellitus (T1D). We examined the functional and anatomical effects of T1D on PVG and studied the details of T1D‐induced remodeling on the PVG structure and function.Methods and ResultsWe used a mouse model of T1D (Akita mouse), immunofluorescence, isolated Langendorff‐perfused hearts, and mathematical simulations to explore the effects of T1D on PVG. Whole‐mount atrial immunofluorescence of choline acetyltransferase and tyrosine hydroxylase labeling showed that sympathetic and parasympathetic somas of the PVG neurons were significantly hypotrophied in T1D hearts versus wild type. Stimulation of PVG in isolated Langendorff‐perfused hearts caused more pronounced P‐P interval prolongation in wild type compared with Akita hearts. Propranolol resulted in a comparable P‐P prolongation in both phenotypes, and atropine led to more pronounced P‐P interval shortening in wild type compared with Akita hearts. Numerical modeling using network simulations revealed that a decrease in the sympathetic and parasympathetic activities of PVG in T1D could explain the experimental results.ConclusionsT1D leads to PVG remodeling with hypotrophy of sympathetic and parasympathetic cell bodies and a concomitant decrease in the PVG sympathetic and parasympathetic activities.

Effect of diabetes and hyaluronidase on the retinal endothelial glycocalyx in mice

We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW = 155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW = 376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.

Inhibition of mTORC1 by ER stress impairs neonatal β-cell expansion and predisposes to diabetes in the Akita mouse.

Unresolved ER stress followed by cell death is recognized as the main cause of a multitude of pathologies including neonatal diabetes. A systematic analysis of the mechanisms of β-cell loss and dysfunction in Akita mice, in which a mutation in the proinsulin gene causes a severe form of permanent neonatal diabetes, showed no increase in β-cell apoptosis throughout life. Surprisingly, we found that the main mechanism leading to β-cell dysfunction is marked impairment of β-cell growth during the early postnatal life due to transient inhibition of mTORC1, which governs postnatal β-cell growth and differentiation. Importantly, restoration of mTORC1 activity in neonate β-cells was sufficient to rescue postnatal β-cell growth, and to improve diabetes. We propose a scenario for the development of permanent neonatal diabetes, possibly also common forms of diabetes, where early-life events inducing ER stress affect β-cell mass expansion due to mTOR inhibition.

Diabetic bladder dysfunction is associated with bladder inflammation triggered through hyperglycemia, not polyuria.

PurposeDiabetes is a grave and progressive condition characterized by debilitating complications. Diabetic bladder dysfunction (DBD) is a very common complication with no specific treatments currently available. Unlike other tissues affected by this disease, the bladder is subjected to two independent insults; 1) polyuria, created by the osmotic effects of glucose in the urine, and 2) hyperglycemia itself. Based on our understanding of inflammation as a major contributor to the underlying organ damage in several other diabetic complications, its presence in the bladder during DBD and the contribution of polyuria and hyperglycemia to its development were assessed.MethodsAwake, restrained cystometry was performed on wild type C57BL/6 mice and diabetic (Akita) mice on a C57BL/6 background at 15 weeks of age. A subgroup of the Akita mice were treated with phlorizin, an inhibitor of sodium-glucose linked transporter types 1 and 2 that prevents glucose reabsorption in the kidney. All groups were assessed for serum glucose, 4-hour voiding totals, and inflammation in the bladder (Evans blue assay).ResultsAkita mice develop cystometrically-defined DBD by 15 weeks of age, as evidenced by an increase in urinary frequency, a decrease in voiding volume, and an increase in post-voiding residual volume. Phlorizin effectively normalized serum glucose in these animals while increasing the urine output. Inflammation in the bladder was present in the diabetic animals at this time point, but not detectable in animals receiving phlorizin.ConclusionInflammation in the bladder of diabetic mice correlates with the development of DBD and is triggered by hyperglycemia, not polyuria.

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2Akita.

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

Abstract P246: Increased Urinary Prorenin and sPRR Excretion in Early Type 1 Diabetes

Activation of the intra-renal, rather than systemic renin angiotensin system (RAS) has been suggested to play a role in the development of diabetic kidney disease. In this study, we examined plasma and urinary prorenin/renin levels and urinary soluble prorenin receptor (sPRR) levels in a murine model of Type 1 diabetes. Male C57BL/6 mice were injected with streptozotocin (STZ) 50mg/kg at 3 months of age and metabolic balance studies conducted at 6 and 10 weeks post-injection. STZ injected mice developed hyperglycemia within 4 weeks of injection (STZ: 335 ± 35 vs control: 145 ± 22 mg/dL) and remained hyperglycemic at 10 weeks. At 10 weeks post-injection, STZ injected mice had significant polyuria (STZ: 20.9 ± 5.9 vs controls 1.8 ± 0.4 ml/day), increased water intake (STZ: 26.2 ± 5.9 vs controls: 6.2 ± 0.9 ml/day) and lower body weight (STZ: 28.3 ± 1.3 vs controls: 23.6 ± 1.4 grams) despite similar food intake. Compared to controls, urinary angiotensinogen, total prorenin/renin and sPRR levels were increased in STZ injected mice; these increases were proportionally greater than the trend in increased urinary microalbumin excretion (Table 1). In particular, urinary sPRR excretion was markedly increased in STZ injected mice compared to controls. In a separate experiment, 10 week old male Akita mice bearing a mutated Ins2 gene, demonstrated a 4.8 fold increase in renal medullary renin mRNA expression and a 1.5-fold increase in renal medullary PRR expression. Taken together, these results demonstrate early activation of the intra-renal RAS in Type 1 diabetes. These changes occur independent of the systemic RAS.

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage- c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes

Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and β cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of β cells in females but not in males.

MitoGamide ameliorates diabetic cardiomyopathy by scavenging mitochondrial dicarbonyls in type 1 diabetic Akita mice

MitoGamide ameliorates diabetic cardiomyopathy by scavenging mitochondrial dicarbonyls in type 1 diabetic Akita mice

Deletion of 12/15-Lipoxygenase Preserves Retinal Neuronal Function in Diabetic Retinopathy

Purpose: Diabetic retinopathy (DR) is a neurovascular complication of diabetes with critical limitations in current treatment due to a lack of neuroprotection. These limitations mandate the necessity for new therapy that considers the neuronal component of DR. The goal of this study is to explore the role of 12/15-lipoxygenase (LO) in diabetes-induced retinal neuronal dysfunction. Methods: 12/15-LO deficient (12/15-LO−/−) mice were crossbred with Ins2Akita/+ Akita) mice, a classical model of DR, to generate a new mouse model with a double knockout (12/15-LO−/−/Akita). The generated mice were subjected to comprehensive electroretinogram (ERG) analyses after 6 months of diabetes. Results: Profound effects of knocking 12/15-LO out on diabetes-induced reduction in visual system’s functionality were observed. First, Scotopic Threshold Response (pSTR) peak heights, which measure the functionality of ganglion cells (GCs), are consistently lower in Akita than in wild type (WT) mice. Knocking 12/15-LO out significantly improved the overall pSTR and significantly restored the loss of the GC marker (Brn3) as compared with Akita mice. Second, diabetes reduced the functionality of cone-bipolar cells by 32%, as assessed by photopic B-Wave (pBW) amplitudes, however, this reduction was significantly improved in 12/15-LO−/−/Akita mice compared with Akita mice (p<0.05, n=9). Lastly, diabetes caused a significant reduction (22%) in cone photoreceptor functionality as indicated by a lower response to natural noise stimuli (NNS) in Akita compared to WT mice (p<0.05, n=8) and this reduction was completely recovered in 12/15-LO−/−/Akita mice (p<0.05, n=9). Conclusion: To our knowledge, this is the first report that elimination of 12/15-LO protects against diabetes-induced visual system dysfunction. The findings may lead to new therapeutic approaches in prevention and treatment of DR. Disclosure M. Wan: None. A. Ibrahim: None. M. El-Shafey: None. K. Elmasry: None. S. Abdulmoneim: None. A. Saul: None. M. Al-Shabrawey: None.

Lactogens Prevent ER Stress-Induced ß-Cell Death and Diabetes

ER stress is highly relevant in the context of diabetes, as inducers of β cell death and dysfunction implicated in the disease - including proinflammatory cytokines and glucolipotoxicity - are known to impair ER function. Previous work has shown that prolactin (Prl) and placental lactogen (PL), which bind to the same receptor (Prl-R), are important for β cell function and proliferation, and enhance survival against cytokines and glucolipotoxicity. Here, we examine the effect of Prl/PL directly on ER stress and the associated unfolded protein response (UPR) in the β cell. ER stress was induced in vitro via tunicamycin in INS1, primary mouse, and primary human β cells, concurrent with Prl/veh treatment. Expression of ER stress and UPR associated genes was measured via qRT-PCR and preliminarily via WB, and insulin-TUNEL co-staining was used to assay for β cell death. As an in vivo model, heterozygous Akita mice - a well-established β cell ER stress model which becomes diabetic by 4 weeks of age - were bred to single-transgenic mice overexpressing PL in the β cell. Prl increases survival of INS1, primary mouse and human β cells against ER stress-mediated cell death in vitro (2.34±0.19% TM vs. 0.73±0.19% TM+Prl, human). Differences in mRNA expression point towards modulation of the PERK/IRE1 UPR arms. Non-Tg Akita mice quickly become and remain hyperglycemic/diabetic, while overexpression of PL in the β cells of these mice significantly improves glycemia in males (663±29mg/dL non-Tg vs. 327±37mg/dL Tg, 12 weeks) and results in normoglycemia in females. In vivo GSIS at 12 weeks shows a trend towards restoration of β cell function in Tg compared to non-Tg Akita mice. Males at this age exhibit improved β cell mass (0.28±0.12mg non-Tg vs. 2.29±0.44mg Tg), with similar trends in β cell proliferation. Taken together, these data demonstrate that Prl/PL can prevent and protect against ER stress in the β cell, in vitro and in vivo. As ER stress is highly relevant to diabetes, modulation of the lactogenic pathway could present a novel therapy for this disease. Disclosure R. Li: None. R.F. Hampton: None. N. Guthalu Kondegowda: None. R. Fenutria: None. R.C. Vasavada: None.

Sclerostin Resistance Protects Bone Mass and Retards the Onset of Metabolic Abnormalities in a Mouse Model of Type 1 Diabetes

Type 1 diabetes (T1D) is characterized by bone fragility and increased risk of fracture. In humans and preclinical models of T1D, serum markers of bone formation are decreased suggesting reduced bone turnover rates. In a preliminary study, we found high circulating levels of the Wnt inhibitor sclerostin (Scl) in T1D patients. To test the potential role of Scl and Wnt in T1D bone disease, we introduced the Scl-resistant Lrp5A214V mutation, associated with high bone mass (HBM), in mice carrying the Ins2Akita mutation (Akita), which causes hypoinsulinemia and hyperglycemia at age 4-5 week. Both Akita and Akita/HBM mutants developed diabetes (non-fasting blood glucose >300 mg/dl), albeit with different onset timing. At 6 weeks, only 30% of Akita/HBM mice had developed hyperglycemia, compared to 90% of Akita mice (n=10); and at 8 weeks only 40% of Akita/HBM were hyperglycemic vs. 90% in the Akita group. Only at 12 weeks was the majority of Akita/HBM mice hyperglycemic, as the Akita group. Furthermore, Akita mice exhibited a significant impairment in glucose tolerance in an intraperitoneal glucose tolerance test relative to Akita/HBM mice at 6 and 8 weeks (p<0.for difference in areas under the curve; n=3-6). Thus, Wnt signaling hyperactivation in Scl resistant mice may slow the onset of T1D, although it does not prevent it. No significant differences in weight and nose-tail length were found in Akita/HBM mutants and controls. Importantly, bone mass was 18% higher in Akita/HBM relative to Akita littermates at 12 weeks (p<0.001; n=3-6), a difference that persisted up until at least 18 weeks of age, despite prolonged hyperglycemia. Bone mass in compound mutants was not different relative to HBM mice. Thus, the metabolic changes caused by T1D do not alter the consequence of Scl resistance and Wnt hyperactivation on bone. In summary, even though we have not yet measured bone strength, Wnt stimulation protects bone mass and may retard the onset of metabolic abnormalities in T1D. Disclosure G. Leanza: None. R. Strollo: None. P. Pozzilli: Research Support; Self; Sanofi. Speaker's Bureau; Self; Eli Lilly and Company. Research Support; Self; Merck Sharp & Dohme Corp.. R. Civitelli: None. N. Napoli: None.

Hyperglycemia Promotes Muscle Atrophy through the WWP1/KLF15 Pathway

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.

Angiotensin-Converting Enzyme 2 Deficiency Increases Translocation of Gut Bacteria by Depletion of Bone Marrow-Derived Circulating Angiogenic Cells

A healthy microbiome is central to good nutrition and efficient metabolism both of which can be altered in diabetes. Disruption of gut-vascular integrity may lead to translocation of microbial antigens and bacteria into the circulation. Bone marrow-derived circulating angiogenic cells (CACs) are essential to maintain vascular integrity. We tested the hypothesis that angiotensin-converting enzyme 2 (ACE2), the key enzyme in the vasoprotective axis of renin-angiotensin system (RAS), acts to regulate the infiltration of bone marrow-derived CACs in the gut and to maintain gut-vascular integrity in diabetes and could influence the systemic translocation of pathogenic antigens and bacteria from the gut. Diabetic ACE2 knockout (ACE2-/y)/C57BL/6-Ins2WT/C96Y (Akita) mice were examined at 9 months after the onset of diabetes and compared to age-matched controls. Beta diversity analysis of the gut microbiome revealed a distinct bacterial profile among the different genotypes (n=9-17 per group, p=0.002). The fecal samples from ACE2-/y-Akita group compared to Akita group were enriched with a great diversity of bacterial taxa that have been reported to contribute to the pathogenesis of diabetes and showed activation of peptidoglycan biosynthesis pathways by metatranscriptome analysis (n=4 per group, p=0.005). Flow cytometry analysis showed that loss of ACE2 led to reduced infiltration of bone marrow-derived CACs (CD45+Flk-1+CD31+) into the gut (n=8-14 per group, p<0.0001), without modifying levels of proinflammatory or immune cell phenotypes. The CAC depletion mediated by ACE2 deficiency was associated with increased levels of gut bacteria in the blood as assessed by SourceTracker analysis (n=9 per group). These data suggested a loss of the protective arm of RAS contributes to the depletion of CACs and disruption of gut-vascular barrier that may promote bacterial translocation in diabetes. Disclosure Y. Duan: None. D. Feng: None. E. Beli: None. S. Li Calzi: None. A.F. Longhini: None. R. Lamendella: None. J.R. Wright: None. T.A. Markel: None. A.G. Obukhov: None. M.B. Grant: None.

NADPH Oxidase Nox5 Aggravates Renal Injury in Akita Mouse Model of Diabetic Nephropathy

Background: Renal reactive oxygen species (ROS) play an important role in mediating kidney injury in diabetes. Increasing evidence suggests that the pro-oxidant enzyme, Nox5 plays a significant role in human diabetic nephropathy (DN). Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional animal models of DN. We examined the role of Nox5 in the insulin deficient diabetic Akita mice model using human inducible transgenic mice that express Nox5 selectively in endothelial cells (VEcad+Nox5+) or in mesangial cells (SM22+Nox5+). We also examined the endogenous expression of Nox5 in a high fat fed rabbit model of kidney disease. Methods: At week 10 mice were culled and kidneys were removed for the assessment of structural damage as well as gene and protein expression of markers of inflammation, fibrosis and oxidative stress. Protein expression of Nox5 and its localization in glomerular cells (endothelial and mesangial cells) were examined in transgenic mice by immunostaining. We also examined expression of pro-fibrotic gene in high fat fed rabbits by next generation sequencing (NGS) and RT-PCR and renal injury by histochemistry. Results: Expression of Nox5 was confirmed in glomerular endothelial and mesangial cells of transgenic mice. Diabetes induced increase in glomerulosclerosis, gene and protein expression of fibronectin and MCP-1 as well as nitrotyrosine were further increase in both diabetic Nox5 transgenic mice. Moreover, increased expression of Nox5 in high fat fed rabbits vs. normal diet fed rabbits was associated with increased expression of fibronectin, CTGF, collagen IV and VCAM-1 as well as increased mesangial expansion in the kidney. Conclusions: These findings in both transgenic mice model (endothelial and mesangial cells) suggest that Nox5 plays a significant role in mediating renal injury in diabetes. Disclosure J.C. Jha: None. A. Dai: None. M.E. Cooper: Advisory Panel; Self; AbbVie Inc.. Research Support; Self; Boehringer Ingelheim GmbH. Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc.. Speaker's Bureau; Self; Eli Lilly and Company. Advisory Panel; Self; Merck Sharp & Dohme Corp., AstraZeneca. Research Support; Self; Novo Nordisk A/S. Advisory Panel; Self; Novartis AG. R.M. Touyz: None. C. Kennedy: Research Support; Self; Prometic Life Sciences Inc.. K. Jandeleit-Dahm: None.

Urocortin-2 Gene Transfer Effectively Treats Type 1 Diabetes in Mice

Type 1 diabetes (T1D) affects 1.3 million U.S. patients. Tight glucose control reduces microvascular complications and adverse cardiovascular events. Insulin therapy is essential but has shortcomings: a) only 1 in 3 patients achieve targeted glucose control; b) aggressive insulin use increases hypoglycemia; c) 25% of T1D patients develop insulin resistance. We previously reported that urocortin-2 (UCn2) gene transfer increases insulin sensitivity and release in mouse models of type 2 diabetes. In the present study, 3m-old male Akita mice with T1D (due to Ins2 mutation) received IV saline, AAV8. Null or AAV8.UCn2 (2x1012 gc/kg). Ten weeks after UCn2 gene transfer we saw normalization of fasting glucose, HbA1c and glucose tolerance (Table). Increased body weight and reduced water intake was seen after UCn2 gene transfer. Hyperinsulinemic euglycemic clamps showed increased insulin sensitivity and skeletal muscle glucose uptake. There was reduced nephropathy and retinopathy and increased survival (Table). Echocardiography showed improved ejection fraction (p<.001), end-systolic dimension (p<.001) and diastolic function (p<.0001). Data acquisition and analyses were blinded. The mechanisms for beneficial effects include increased insulin release and sensitivity. Thus, UCn2 gene transfer may be a viable therapy both for new onset T1D with detectable endogenous insulin, and for later stage T1D to reduce exogenous insulin needs. UCn2 Gene Transfer in Akita Mice (Type 1 Diabetes)Saline or AAV8.Null (n)AAV8.UCn2 (n)pPlasma UCn2 (ng/mL)1±.2 (14)12±.9 (14)<.0001Urine Output (ml/hr).43±.06 (8).24±.06 (8).0412hr Fasting Glucose (mg/dL)272±25 (8)106±12 (8)<.0001HbA1c (%)8.2±.4 (5)5.5±.4 (5)<.002Glucose Tolerance Test (AUC)3128±215 (8)2062±286 (8)<.0001Insulin Release 2h post gluc (ng/ml).15±.01 (8).29±.4 (8).004Glucose Infusion Rate (mg/kg/min)12±2.89 (6)33.6±5.3 (5).001Vast Lat Glu Uptake (µM/m/100g)8.2±.4 (7)23.2±2.6 (6)<.0001Glomerulus Expansion Score1.7±.04 (8)1.5±.04 (8).01Urine Albumin/Creatinine (3hr)28±3 (8)17±3 (8).01Retinal Vascular Leak (%)43±1 (5)19±5 (4).001200-day Survival (%)0 (17 of 17 died)82 (3 of 17 died)<.0001Mean ± SE; p from Student’s t-test (unpaired, 2 tails); AAV8, Adeno-Associated Virus Type 8 Disclosure M. Gao: None. N. Lai: None. D. Giamouridis: None. T.Y. Guo: None. B. Xia: None. Y. Kim: None. M.V. Estrada: None. V. Nguyen Huu: None. D. Skowronska-Krawczyk: None. H. Hammond: Stock/Shareholder; Self; Renova Therapeutics.

Impaired Nociception in the Diabetic Ins2+/Akita Mouse.

The mechanisms responsible for painful and insensate diabetic neuropathy are not completely understood. Here, we have investigated sensory neuropathy in the Ins2+/Akita mouse, a hereditary model of diabetes. Akita mice become diabetic soon after weaning, and we show that this is accompanied by an impaired mechanical and thermal nociception and a significant loss of intraepidermal nerve fibers. Electrophysiological investigations of skin-nerve preparations identified a reduced rate of action potential discharge in Ins2+/Akita mechanonociceptors compared with wild-type littermates, whereas the function of low-threshold A-fibers was essentially intact. Studies of isolated sensory neurons demonstrated a markedly reduced heat responsiveness in Ins2+/Akita dorsal root ganglion (DRG) neurons, but a mostly unchanged function of cold-sensitive neurons. Restoration of normal glucose control by islet transplantation produced a rapid recovery of nociception, which occurred before normoglycemia had been achieved. Islet transplantation also restored Ins2+/Akita intraepidermal nerve fiber density to the same level as wild-type mice, indicating that restored insulin production can reverse both sensory and anatomical abnormalities of diabetic neuropathy in mice. The reduced rate of action potential discharge in nociceptive fibers and the impaired heat responsiveness of Ins2+/Akita DRG neurons suggest that ionic sensory transduction and transmission mechanisms are modified by diabetes.

Lactobacillus plantarum reverse diabetes-induced Fmo3 and ICAM expression in mice through enteric dysbiosis-related c-Jun NH2-terminal kinase pathways.

Diabetes mellitus (DM) is characterized by increased fatality associated with the atherogenetic process. Circulating trimethylamine-N-oxide (TMAO) levels are closely associated with atherosclerosis. The flavin mono-oxygenase family (Fmo) members oxidize trimethylamine (TMA) to TMAO. The effect and the regulatory mechanism of intestinal microflora on diabetes-induced Fmo3 and intercellular adhesion molecule (ICAM) expression were examined in streptozotocin-induced diabetic mice (STZDM) and Akita mice (C57BL/6J-Ins2Akita). STZDM-JNK1-/- and Ins2Akita-JNK1-/- mice were produced and used to study the role of pJNK in the regulatory mechanisms. Diabetic mice exhibited decreased Lactobacilli growth and reactive oxygen species (ROS) production in the intestinal mucosa; increased levels of pJNK and iNOS proteins in the intestinal mucosa; increased levels of serum nitrate, IL-1β, and TNF-α expression in Kupffer cells; increased Fmo3 expression in the liver; and increased ICAM expression in the aorta. Reversal of diabetes-induced enteric dysbiosis by prebiotic (FOS) or probiotic (dead L. plantarum) treatment decreased diabetes-induced pJNK and iNOS expression in the intestine, Fmo3 expression in the liver, IL-1β expression in Kupffer cells, and ICAM expression in the aorta and liver. Ins2Akita-JNK1-/- and STZDM-JNK1-/- mice demonstrated decreased levels of serum NO, IL-1β expression in Kupffer cells, Fmo3 expression in the liver, and ICAM expression in the aorta. GF mice cohoused with DM mice demonstrated an increase in ICAM expression in the liver. In conclusion, diabetes induced the expression of both Fmo3 and ICAM expression and possible vascular impairment through enteric dysbiosis. Diabetes-induced Fmo3 and ICAM expression could be reversed by pJNK inhibition or by correcting enteric dysbiosis.

Evidence of extensive atherosclerosis, coronary artery disease and myocardial infarction in the ApoE−/−:Ins2+/Akita mouse fed a western diet

Background and aimsDiabetic patients with no history of cardiac infarction have a prevalence of coronary atherosclerosis and a risk of heart attack equivalent to euglycemic patients who have coronary atherosclerosis and have suffered a prior myocardial infarction. Although several murine models of diabetes have been established, none of these show indications of cardiac events. In an attempt to establish a diabetic mouse model with coronary atherosclerosis and myocardial injury, we have fed hyperglycemic ApoE−/−:Ins2+/Akita mice a western diet to enhance the dyslipidemic phenotype. MethodsFive-week-old ApoE−/−:Ins2+/Akita mice and ApoE−/− controls were fed a diet, 0.15% cholesterol and 21% anhydrous milk lipids, until 25 weeks of age. Changes in lifespan, clinical and metabolic parameters were evaluated as well as atherosclerosis and heart injury. ResultsIn comparison to male ApoE−/−, male ApoE−/−:Ins2+/Akita mice presented with chronic hyperglycemia (30.8 ± 1.2 mM vs. 9.3 ± 0.5 mM) accompanied by extremely high levels of total plasma cholesterol (49.3 ± 6.3 mM vs. 30.1 ± 1.5 mM) and triglycerides (11.6 ± 1.7 mM vs. 2.36 ± 0.18 mM). These mice have atherosclerosis at multiple vascular sites, including aortic sinus, ascending and descending aorta, brachiocephalic artery and coronary arteries. In addition, myocardial infarcts and a significant reduction of the lifespan (close to 20% of survival vs. other groups) were observed. Distinctively, both strains of female mice presented a parallel increase in plasma lipids, atherosclerosis, and no effects on mortality. ConclusionsWe have established a diabetic mouse model, the western-diet-fed male ApoE−/−:Ins2+/Akita mouse, with profound cardiovascular disease involving extensive atherosclerosis, coronary artery disease and myocardial infarct resulting in shortened lifespan.