Transgenic overexpression of leptin rescues insulin resistance and diabetes in a mouse model of lipoatrophic diabetes

Lipoatrophic diabetes is caused by a deficiency of adipose tissue and is characterized by severe insulin resistance, hypoleptinemia, and hyperphagia. The A-ZIP/F-1 mouse (A-ZIPTg/+) is a model of severe lipoatrophic diabetes and is insulin resistant, hypoleptinemic, hyperphagic, and shows severe hepatic steatosis. We have also produced transgenic "skinny" mice that have hepatic overexpression of leptin (LepTg/+) and no adipocyte triglyceride stores, and are hypophagic and show increased insulin sensitivity. To explore the pathophysiological and therapeutic roles of leptin in lipoatrophic diabetes, we crossed LepTg/+ and A-ZIPTg/+ mice, producing doubly transgenic mice (LepTg/+:A-ZIPTg/+) virtually lacking adipose tissue but having greatly elevated leptin levels. The LepTg/+:A-ZIPTg/+ mice were hypophagic and showed improved hepatic steatosis. Glucose and insulin tolerance tests revealed increased insulin sensitivity, comparable to LepTg/+ mice. These effects were stable over at least 6 months of age. Pair-feeding the A-ZIPTg/+ mice to the amount of food consumed by LepTg/+:A-ZIPTg/+ mice did not improve their insulin resistance, diabetes, or hepatic steatosis, demonstrating that the beneficial effects of leptin were not due to the decreased food intake. Continuous leptin administration that elevates plasma leptin concentrations to those of LepTg/+:A-ZIPTg/+ mice also effectively improved hepatic steatosis and the disorder of glucose and lipid metabolism in A-ZIP/F-1 mice. These data demonstrate that leptin can improve the insulin resistance and diabetes of a mouse model of severe lipoatrophic diabetes, suggesting that leptin may be therapeutically useful in the long-term treatment of lipoatrophic diabetes.     

Transgenic mouse models of type I diabetes

Transgenic mouse technology has gained recognition as an important tool for examining many fundamental biological questions in vivo. Recently, transgenic mouse techniques have been applied to the study of type I (insulin-dependent) diabetes. These studies have been particularly informative in elucidating 1) mechanisms whereby immune tolerance is maintained to antigens on rare specialized cells such as the pancreatic beta-cell, 2) disease susceptibility and resistance genes, and 3) potentially important immune effector mechanisms. In this article, we discuss these studies, their impact on understanding of the pathogenesis of type I diabetes, and the potential of the transgenic mouse approach for future research.     

Transgenic rats carrying the mouse renin gene--morphological characterization of a low-renin hypertension model.

Transgenic rats [TGR; strain name TGR(mRen2)27] harboring the mouse Ren-2 renin gene have been recently generated as a model for the study of primary hypertension that offers the advantage of a clearly-defined genetic alteration. Expression of the mouse Ren-2 gene causes severe hypertension (200 to 260 mm Hg) which is responsive to converting enzyme inhibitors. Compared to control transgene-negative littermates, plasma renin and angiotensin II values are lowered in TGR, whereas plasma prorenin values are strongly elevated. The adrenal gland in TGR shows marked overexpression of mouse renin messenger RNA; in situ hybridization using a 35S-labelled mouse-renin RNA probe reveals that enhanced renin expression is mainly localized to cells of the zona glomerulosa and outer zona fasciculata. Immunohistochemically, renin protein in the TGR adrenal gland is stored in larger quantities than in controls. Adrenal transgene expression probably accounts for most of the elevated plasma prorenin level in TGR, since bilateral adrenalectomy (ADX) causes a significant decrease in prorenin level (318 +/- 79 ng angiotensin I/ml/hr before ADX to 70 +/- 43 ng 4 days after ADX, P less than 0.0005). In the kidney, renin synthesis is almost completely suppressed in TGR. In situ hybridization demonstrates that few juxtaglomerular afferent arterioles express renin. Immunohistochemically, the TGR kidney shows significantly reduced renin and angiotensin II immunoreactivity at the afferent arteriole. Ultrastructural analysis of the afferent arteriolar wall frequently shows the complete absence of renin secretory granules since the granular cells are mostly converted into smooth muscle cells. Beginning at an age of approximately four to six months, TGR develop hypertension-related alterations and pathological lesions in various tissues. In the kidney, the wall thickness of arterioles and arteries is strongly increased, and glomerular lesions including different stages of sclerosis are observed. The thoracic aorta displays a considerable increase in tunica media thickness due to both myocyte hypertrophy and interstitial fibrosis. Coronary arteries and arterioles of the heart are thickened and perivascular fibrosis is observed. The data show that TGR(mRen2)27 transgenic rats display all typical characteristics of hypertensive pathology, making them an interesting model for therapeutic interventions. The fact that these changes occur in animals with a single gene difference to normotensive rats makes them a particularly suitable model for studies on gene-related hypertensive processes.     

An experimental therapy to improve skeletal growth and prevent bone loss in a mouse model overexpressing IL-6

Summary

Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory disease. Presently, no treatment regimens are available for these defects in juvenile diseases. We identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in a mouse model overexpressing IL-6.

Introduction

Premature osteoporosis and stunted growth are common complications of childhood chronic inflammatory diseases and have a significant impact on patients’ quality of life. Presently, no treatment regimens are available for these defects in juvenile diseases. To test a new therapeutic approach, we used growing mice overexpressing the pro-inflammatory cytokine IL-6 (TG), which show a generalized bone loss and stunted growth.

Methods

Since TG mice present increased bone resorption and impaired bone formation, we tested a combined therapy with the antiresorptive modified osteoprotegerin, Fc-OPG, and the anabolic PTH. We injected TG mice with Fc-OPG once at the 4th day of life and with hPTH(1–34) everyday from the 16th to the 30th day of age.

Results

A complete prevention of growth and bone defects was observed in treated mice due to normalization of osteoclast and osteoblast parameters. Re-establishment of normal bone turnover was confirmed by RT-PCR analysis and by in vitro experiments that revealed the full rescue of osteoclast and osteoblast functions. The phenotypic recovery of TG mice was due to the sequential treatment, because TG mice treated with Fc-OPG or hPTH alone showed an increase of body weight, tibia length, and bone volume to intermediate levels between those observed in vehicle-treated WT and TG mice.

Conclusions

Our results identified the sequential Fc-OPG/hPTH treatment as an experimental therapy that improves the skeletal growth and prevents the bone loss in IL-6 overexpressing mice, thus providing the proof of principle for a therapeutic approach to correct these defects in juvenile inflammatory diseases.     

Myostatin inhibition prevents diabetes and hyperphagia in a mouse model of lipodystrophy

Lipodystrophies are characterized by a loss of white adipose tissue, which causes ectopic lipid deposition, peripheral insulin resistance, reduced adipokine levels, and increased food intake (hyperphagia). The growth factor myostatin (MSTN) negatively regulates skeletal muscle growth, and mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. MSTN inhibition may therefore be efficacious in ameliorating diabetes. To test this hypothesis, we inhibited MSTN signaling in a diabetic model of generalized lipodystrophy to analyze its effects on glucose metabolism separate from effects on adipose mass. A-ZIP/F1 lipodystrophic mice were crossed to mice expressing a dominant-negative MSTN receptor (activin receptor type IIB) in muscle. MSTN inhibition in A-ZIP/F1 mice reduced blood glucose, serum insulin, triglyceride levels, and the rate of triglyceride synthesis, and improved insulin sensitivity. Unexpectedly, hyperphagia was normalized by MSTN inhibition in muscle. Blood glucose and hyperphagia were reduced in double mutants independent of the adipokine leptin. These results show that the effect of MSTN inhibition on insulin sensitivity is not secondary to an effect on adipose mass and that MSTN inhibition may be an effective treatment for diabetes. These results further suggest that muscle may play a heretofore unappreciated role in regulating food intake.     

Angiotensin II type 1 receptor blockade improves beta-cell function and glucose tolerance in a mouse model of type 2 diabetes

We identified an angiotensin-generating system in pancreatic islets and found that exogenously administered angiotensin II, after binding to its receptors (angiotensin II type 1 receptor [AT1R]), inhibits insulin release in a manner associated with decreased islet blood flow and (pro)insulin biosynthesis. The present study tested the hypothesis that there is a change in AT1R expression in the pancreatic islets of the obesity-induced type 2 diabetes model, the db/db mouse, which enables endogenous levels of angiotensin II to impair islet function. Islets from 10-week-old db/db and control mice were isolated and investigated. In addition, the AT1R antagonist losartan was administered orally to 4-week-old db/db mice for an 8-week period. We found that AT1R mRNA was upregulated markedly in db/db islets and double immunolabeling confirmed that the AT1R was localized to beta-cells. Losartan selectively improved glucose-induced insulin release and (pro)insulin biosynthesis in db/db islets. Oral losartan treatment delayed the onset of diabetes, and reduced hyperglycemia and glucose intolerance in db/db mice, but did not affect the insulin sensitivity of peripheral tissues. The present findings indicate that AT1R antagonism improves beta-cell function and glucose tolerance in young type 2 diabetic mice. Whether islet AT1R activation plays a role in the pathogenesis of human type 2 diabetes remains to be determined.     

Successful reversal of diabetes by single donor isologous islet transplantation in a mouse model

A method for isolating mouse islets which consistently gives a high yield with good purity is described. Using a bovine serum albumin gradient, the mean yield of islets per pancreas is 425 (SEM ± 15) with a consistent purity of over 90%. Single donor to single recipient of islets transplanted under the renal capsule restores normoglycemia in the diabetic recipients within 2 to 5 days of transplantation.     

A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site

Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.     

Bone formation is impaired in a model of type 1 diabetes

The effects of type 1 diabetes on de novo bone formation during tibial distraction osteogenesis (DO) and on intact trabecular and cortical bone were studied using nonobese diabetic (NOD) mice and comparably aged nondiabetic NOD mice. Diabetic mice received treatment with insulin, vehicle, or no treatment during a 14-day DO procedure. Distracted tibiae were analyzed radiographically, histologically, and by microcomputed tomography (microCT). Contralateral tibiae were analyzed using microCT. Serum levels of insulin, osteocalcin, and cross-linked C-telopeptide of type I collagen were measured. Total new bone in the DO gap was reduced histologically (P < or = 0.001) and radiographically (P < or = 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < or = 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by microCT and mechanical testing demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals, which were prevented by insulin treatment. These studies demonstrate that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration.     

New mouse model to study islet transplantation in insulin-dependent diabetes mellitus

BACKGROUND: Islet transplantation studies with diabetic rodents frequently use treatment with diabetogens such as alloxan or streptozotocin to render hosts hyperglycemic. These chemicals produce unwanted toxic side effects, which complicate interpretations of damage produced by hyperglycemia versus direct toxin-induced damage. A mouse that spontaneously developed insulin-sensitive diabetes without beta-cell autoimmunity would provide an excellent vehicle for testing beta-cell replacement protocols. The Ins2Akita mutation disrupts normal insulin processing and causes a failure in secretion of mature insulins, which results in the early development of hyperglycemia. This report examines the insulin sensitivity of mice that carry Ins2Akita and their responsiveness to engraftment with syngeneic pancreatic islets. METHODS: Ten-week-old C57BL/6J-Ins2Akita/+ males were given 1 unit of insulin to determine insulin sensitivity. Also, 10-week-old, hyperglycemic B6-Ins2Akita/+ received either 400 islets isolated from syngeneic C57BL/6J males (n=7) or from allogeneic BALB/cJ males (n=5) under the renal capsule. These mice were followed for 8 weeks after engraftment or until remission of euglycemia. Nephrectomy of the graft-containing kidney was performed on mice that remained euglycemic. These mice were then followed for 2 weeks for return of hyperglycemia. RESULTS: B6-Ins2Akita/+ mice are insulin responsive. Insulin treatment of hyperglycemic B6-Ins2Akita/+ males significantly lowered blood glucose values within 1 hr. In addition, B6-Ins2Akita/+ recipients of syngeneic islet grafts reversed their diabetic state in less than 72 hr. These islet-engrafted mice remained normoglycemic until removal of the graft-containing kidney. Removal of the graft resulted in a return to hyperglycemia. Mice that received allogeneic grafts efficiently rejected the graft. CONCLUSIONS: Our data support the hypothesis that B6-Ins2Akita/+ mice are insulin sensitive and provide an excellent model for islet transplantation studies. In addition, the reduced beta-cell mass and the absence of beta-cell autoimmunity, coupled to the fact that these mice also reject allografts, suggest that these mice may be useful for a variety of other applications, including testing functionality of human islets prepared for transplantation and perhaps also for exploring beta-cell restorative therapy using pancreatic islet stem cells.     

A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.     

应用Cuff技术建立小鼠异位心脏移植模型

目的建立简单易行、稳定可靠的小鼠颈部异位心脏移植模型。方法自制血管套管，运用Cuff技术吻合移植心的动、静脉。结果正式实验25次，成功23例，供心缺血时间均少于30min。结论该方法明显降低了手术难度，具有手术时间短，创伤小，成功率高的优点，是一种简单实用的方法。     

Genetic analysis of late-onset type 2 diabetes in a mouse model of human complex trait.

Type 2 diabetes is a complex trait with both genes and environmental factors contributing to susceptibility. Except for rare subtypes with monogenic inheritance, the genetic basis of type 2 diabetes is unknown because of the complex and heterogeneous nature of the disease. By using the NSY mouse, an inbred mouse model of type 2 diabetes, we genetically dissected late-onset type 2 diabetes and demonstrated age-dependent changes in the genetic control of type 2 diabetes as well as polygenic inheritance. Three major loci (Nidd1nsy, Nidd2nsy, Nidd3nsy) were mapped on mouse chromosomes (Chr) 11, 14, and 6, respectively. The existence of a fourth locus (Nidd4nsy) with an age-dependent effect was suggested by longitudinal, but not cross-sectional, analysis of linkage data. Nidd1nsy and Nidd4nsy appear to affect insulin secretion, whereas Nidd2nsy and Nidd3nsy appear to affect insulin sensitivity. A locus on Chr 6 was significantly linked to epididymal fat weight. A candidate disease gene (Tcf2) on Chr 11, encoding hepatic nuclear factor-1beta, was shown to have a rare sequence variant in the DNA binding domain in the model. The mouse model we used will serve as a useful model for future studies on the etiology of late-onset polygenic type 2 diabetes in humans.     

Maresin 1 attenuates neuroinflammation in a mouse model of perioperative neurocognitive disorders

Background

Resolution of inflammation is an active and dynamic process after surgery. Maresin 1 (MaR1) is one of a growing number of specialised pro-resolving lipids biosynthesised by macrophages that regulates acute inflammation. We investigated the effects of MaR1 on postoperative neuroinflammation, macrophage activity, and cognitive function in mice.