Altered REDD1, myostatin, and Akt/mTOR/FoxO/MAPK signaling in streptozotocin-induced diabetic muscle atrophy

Type 1 diabetes, if poorly controlled, leads to skeletal muscle atrophy, decreasing the quality of life. We aimed to search highly responsive genes in diabetic muscle atrophy in a common diabetes model and to further characterize associated signaling pathways. Mice were killed 1, 3, or 5 wk after streptozotocin or control. Gene expression of calf muscles was analyzed using microarray and protein signaling with Western blotting. We identified translational repressor protein REDD1 (regulated in development and DNA damage responses) that increased seven- to eightfold and was associated with muscle atrophy in diabetes. The diabetes-induced increase in REDD1 was confirmed at the protein level. This result was accompanied by the increased gene expression of DNA damage/repair pathways and decreased expression in ATP production pathways. Concomitantly, increased phosphorylation of AMPK and dephosphorylation of the Akt/mTOR/S6K1/FoxO pathway of proteins were observed together with increased protein ubiquitination. These changes were especially evident during the first 3 wk, along with the strong decrease in muscle mass. Diabetes also induced an increase in myostatin protein and decreased MAPK signaling. These, together with decreased serum insulin and increased serum glucose, remained altered throughout the 5-wk period. In conclusion, diabetic myopathy induced by streptozotocin led to alteration of multiple signaling pathways. Of those, increased REDD1 and myostatin together with decreased Akt/mTOR/FoxO signaling are associated with diabetic muscle atrophy. The increased REDD1 and decreased Akt/mTOR/FoxO signaling followed a similar time course and thus may be explained, in part, by increased expression of genes in DNA damage/repair and possibly also decrease in ATP-production pathways.     

Cystathionine γ-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine γ-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth^−/−) mice as an animal model of cystathioninemia/cystathioninuria. Cth^−/− mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth^−/− mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth^−/− hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth^−/− mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine β-synthase (Cbs^−/−). Because Cbs^−/− mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie''s method, which detects hypermethioninemia.     

The DNA damage checkpoint protein ATM promotes hepatocellular apoptosis and fibrosis in a mouse model of non-alcoholic fatty liver disease

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting from excess dietary fat contribute to liver disease by causing DNA damage and apoptotic cell death, and tested this by investigating the effects of feeding mice high fat or standard diets for 8 weeks. High fat diet feeding resulted in increased hepatic H₂O₂, superoxide production, and expression of oxidative stress response genes, confirming that the high fat diet induced hepatic oxidative stress. High fat diet feeding also increased hepatic steatosis, hepatitis and DNA damage as exemplified by an increase in the percentage of 8-hydroxyguanosine (8-OHG) positive hepatocytes in high fat diet fed mice. Consistent with reports that the DNA damage checkpoint kinase Ataxia Telangiectasia Mutated (ATM) is activated by oxidative stress, ATM phosphorylation was induced in the livers of wild type mice following high fat diet feeding. We therefore examined the effects of high fat diet feeding in Atm-deficient mice. The prevalence of apoptosis and expression of the pro-apoptotic factor PUMA were significantly reduced in Atm-deficient mice fed the high fat diet when compared with wild type controls. Furthermore, high fat diet fed Atm^−/− mice had significantly less hepatic fibrosis than Atm^+/+ or Atm^+/− mice fed the same diet. Together, these data demonstrate a prominent role for the ATM pathway in the response to hepatic fat accumulation and link ATM activation to fatty liver-induced steatoapoptosis and fibrosis, key features of NAFLD progression.     

Hyperplasia of Pancreatic Beta Cells and Improved Glucose Tolerance in Mice Deficient in the FXYD2 Subunit of Na,K-ATPase

Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2^−/− mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2^−/− mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2^−/− mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.     

Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.KEY WORDS: Skeletal muscle, Muscle atrophy pathophysiology, TGF-β signaling, Myostatin, Denervation atrophy     

Losartan reduces oxidative damage to renal DNA and conserves plasma antioxidant capacity in diabetic rats

Increased reactive oxygen species (ROS) levels produced by hyperglycemia and angiotensin-II (AT-II) are considered among the pathogenic factors in the malignant transformation of diabetic renal cells. We aimed to investigate the potential role of AT-II in the increased cancer risk seen in diabetes; measuring oxidative damage to renal DNA and protective antioxidant defenses, including adiponectin (Adp) and plasma antioxidant capacity by the Ferric Reducing Ability of Plasma (FRAP) method. In the kidney of streptozotocin (STZ)-induced (55 mg/kg) diabetic rats either treated or not treated for 3 weeks with losartan, an AT-II type 1 receptor antagonist (20 mg/kg/day); we measured 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels, as an index of oxidative DNA damage, circulating Adp and FRAP. Diabetic rats showed significantly higher 8-oxodGuo levels in renal DNA (8.48 ± 0.98 × 10⁻⁶ dG, mean ± SEM n = 11) than normoglycemic ones (1.18 ± 0.04 × 10⁻⁶ dG, mean ± SEM, n=7) and lower plasma Adp and FRAP levels in comparison to normoglycemics. The treatment of diabetic rats with losartan significantly (P < 0.01) reduced 8-oxodGuo levels (5.4 ± 0.58 × 10⁻⁶ dG, mean ± SEM n=9) in renal DNA and conserved FRAP values. Moreover, an inverse correlation was found between 8-oxodGuo in kidney DNA and circulating Adp levels in normoglycemic and diabetic rats. Losartan treatment preserves FRAP levels, reduces DNA oxidative injury and thus the carcinogenesis risk. Furthermore, our results indicate that Adp plasma levels are a further marker of oxidative injury to the kidney and confirm that it is an important part of the plasma antioxidant defense.     

Lack of REDD1 reduces whole body glucose and insulin tolerance,and impairs skeletal muscle insulin signaling

A lack of the REDD1 promotes dysregulated growth signaling, though little has been established with respect to the metabolic role of REDD1. Therefore, the goal of this study was to determine the role of REDD1 on glucose and insulin tolerance, as well as insulin stimulated growth signaling pathway activation in skeletal muscle. First, intraperitoneal (IP) injection of glucose or insulin were administered to REDD1 wildtype (WT) versus knockout (KO) mice to examine changes in blood glucose over time. Next, alterations in skeletal muscle insulin (IRS-1, Akt, ERK 1/2) and growth (4E-BP1, S6K1, REDD1) signaling intermediates were determined before and after IP insulin treatment (10 min). REDD1 KO mice were both glucose and insulin intolerant when compared to WT mice, evident by higher circulating blood glucose concentrations and a greater area under the curve following IP injections of glucose or insulin. While the REDD1 KO exhibited significant though blunted insulin-stimulated increases (p < 0.05) in Akt S473 and T308 phosphorylation versus the WT mice, acute insulin treatment has no effect (p < 0.05) on REDD1 KO skeletal muscle 4E-BP1 T37/46, S6K1 T389, IRS-1 Y1222, and ERK 1/2 T202/Y204 phosphorylation versus the WT mice. Collectively, these novel data suggest that REDD1 has a more distinct role in whole body and skeletal muscle metabolism and insulin action than previously thought.     

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.     

Tocotrienol-rich fraction supplementation reduces hyperglycemia-induced skeletal muscle damage through regulation of insulin signaling and oxidative stress in type 2 diabetic mice

Chronic hyperglycemia induces impairment of muscle growth and development of diabetes mellitus (DM). Since skeletal muscle is the major site for disposal of ingested glucose, impaired glucose metabolism causes imbalance between protein synthesis and degradation which adversely affects physical mobility.In this study, we investigated the effect of tocotrienol-rich fraction (TRF) supplementation on skeletal muscle damage in diabetic mice. Diabetes was induced by a high-fat diet with streptozotocin (STZ) injection (100 mg/kg) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose levels≥250 mg/dl), normal control (CON) and diabetic control (DMC) groups were administrated with olive oil, while TRF treatment groups were administrated with TRF (dissolved in olive oil) at low dose (100 mg/kg BW, LT) or high dose (300 mg/kg BW, HT) by oral gavage for 12 weeks.TRF supplementation ameliorated muscle atrophy, plasma insulin concentration and homeostatic model assessment estimated insulin resistance in diabetic mice. Moreover, TRF treatment up-regulated IRS-1 and Akt levels accompanied by increased translocation of GLUT4. Furthermore, TRF increased mitochondrial biogenesis by activating SIRT1, SIRT3 and AMPK in diabetic skeletal muscle. These changes were in part mechanistically explained by reduced levels of skeletal muscle proteins related to oxidative stress (4-hydroxynonenal, protein carbonyls, Nrf2 and HO-1), inflammation (NFkB, MCP-1, IL-6 and TNF-α), and apoptosis (Bax, Bcl₂ and caspase-3) in diabetic mice. Taken together, these results suggest that TRF might be useful as a beneficial nutraceutical to prevent skeletal muscle atrophy associated with diabetes by regulating insulin signaling via AMPK/SIRT1/PGC1α pathways in type 2 diabetic mice.     

Folate Deficiency Provides Protection against Colon Carcinogenesis in DNA Polymerase β Haploinsufficient Mice

Aging and DNA polymerase β deficiency (β-pol^+/−) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency (FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in β-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in β-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in β-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD β-pol haploinsufficient mice.     

Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects

Telomeres are chromosome end structures and are essential for maintenance of genome stability. Highly repetitive telomere sequences appear to be susceptible to oxidative stress-induced damage. Oxidation may therefore have a severe impact on telomere integrity and function. A wide spectrum of oxidative pyrimidine-derivatives has been reported, including thymine glycol (Tg), that are primarily removed by a DNA glycosylase, Endonuclease III-like protein 1 (Nth1). Here, we investigate the effect of Nth1 deficiency on telomere integrity in mice. Nth1 null (Nth1^−/−) mouse tissues and primary MEFs harbor higher levels of Endonuclease III-sensitive DNA lesions at telomeric repeats, in comparison to a non-telomeric locus. Furthermore, oxidative DNA damage induced by acute exposure to an oxidant is repaired slowly at telomeres in Nth1^−/− MEFs. Although telomere length is not affected in the hematopoietic tissues of Nth1^−/− adult mice, telomeres suffer from attrition and increased recombination and DNA damage foci formation in Nth1^−/− bone marrow cells that are stimulated ex vivo in the presence of 20% oxygen. Nth1 deficiency also enhances telomere fragility in mice. Lastly, in a telomerase null background, Nth1^−/− bone marrow cells undergo severe telomere loss at some chromosome ends and cell apoptosis upon replicative stress. These results suggest that Nth1 plays an important role in telomere maintenance and base repair against oxidative stress-induced base modifications. The fact that telomerase deficiency can exacerbate telomere shortening in Nth1 deficient mouse cells supports that base excision repair cooperates with telomerase to maintain telomere integrity.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

Phosphofructo-1-Kinase Deficiency Leads to a Severe Cardiac and Hematological Disorder in Addition to Skeletal Muscle Glycogenosis

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm^−/−). Here, we show that Pfkm^−/− mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm^−/− mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm^−/− mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm^−/− mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies.     

Genetic Ablation of Calcium-independent Phospholipase A2γ Prevents Obesity and Insulin Resistance during High Fat Feeding by Mitochondrial Uncoupling and Increased Adipocyte Fatty Acid Oxidation

Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A₂γ (iPLA₂γ^−/−) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA₂γ^+/+ mice after high fat feeding. Notably, iPLA₂γ^−/− mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA₂γ^−/− mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA₂γ^−/− mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA₂γ^−/− mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA₂γ^−/− mouse. Collectively, these results identify iPLA₂γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome.     

Diabetic HDL Is Dysfunctional in Stimulating Endothelial Cell Migration and Proliferation Due to Down Regulation of SR-BI Expression

Background

Diabetic HDL had diminished capacity to stimulate endothelial cell (EC) proliferation, migration, and adhesion to extracellular matrix. The mechanism of such dysfunction is poorly understood and we therefore sought to determine the mechanistic features of diabetic HDL dysfunction.

Methodology/Principal Findings

We found that the dysfunction of diabetic HDL on human umbilical vein endothelial cells (HUVECs) was associated with the down regulation of the HDL receptor protein, SR-BI. Akt-phosphorylation in HUVECs was induced in a biphasic manner by normal HDL. While diabetic HDL induced Akt phosphorylation normally after 20 minutes, the phosphorylation observed 24 hours after diabetic HDL treatment was reduced. To determine the role of SR-BI down regulation on diminished EC responses of diabetic HDL, Mouse aortic endothelial cells (MAECs) were isolated from wild type and SR-BI (−/−) mice, and treated with normal and diabetic HDL. The proliferative and migratory effects of normal HDL on wild type MAECs were greatly diminished in SR-BI (−/−) cells. In contrast, response to diabetic HDL was impaired in both types suggesting diminished effectiveness of diabetic HDL on EC proliferation and migration might be due to the down regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.

Conclusions/Significance

Diabetic HDL was dysfunctional in promoting EC proliferation, migration, and adhesion to matrix which was associated with the down-regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.     

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr^−/− mice reconstituted with Akt1^−/− fetal liver cells (Akt1^−/−→Ldlr^−/−) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr^−/−). In contrast, Akt2^−/−→Ldlr^−/− mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr^−/− mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2^−/−→Ldlr^−/− mice had smaller aortic lesions compared with WT→Ldlr^−/− and Akt1^−/−→Ldlr^−/− mice. Importantly, Akt2^−/−→Ldlr^−/− mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6C^hi and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2^−/− mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.     

Global Renal Gene Expression Profiling Analysis in B2-Kinin Receptor Null Mice: Impact of Diabetes

Diabetic nephropathy (DN), the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B₂-receptors (B₂R) in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B₂R knockout (B₂R^−/−) mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER) were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B₂R^−/−D null mice had a significantly decreased AER levels compared to wild type B₂R^+/+D mice (P = 0.0005). Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B₂R^+/+C, B₂R^+/+D, B₂R^−/−C and B₂R^−/−D) highlighted the role of several altered pathological pathways in response to disruption of B₂R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.     

Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD?/?) Mouse to Compensate a Defective Fatty Acid β-Oxidation

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD^−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD^−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD^−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD^−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD^−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.