Leprdb/db Mice with Senescence Marker Protein-30 Knockout (Leprdb/dbSmp30Y/?) Exhibit Increases in Small Dense-LDL and Severe Fatty Liver Despite Being Fed a Standard Diet

Background/Aims

The senescence marker protein-30 (SMP30) is a 34 kDa protein originally identified in rat liver that shows decreased levels with age. Several functional studies using SMP30 knockout (Smp30^Y/−) mice established that SMP30 functions as an antioxidant and protects against apoptosis. To address the potential role of SMP30 in nonalcoholic fatty liver disease (NAFLD) pathogenesis, we established Smp30^Y/− mice on a Lepr^db/db background (Lepr^db/dbSmp30^Y/− mice).

Research Design/Principal Findings

Male Lepr^db/dbSmp30^Y/− mice were fed a standard diet (340 kcal/100 g, fat 5.6%) for 16 weeks whereupon the lipid/lipoprotein profiles, hepatic expression of genes related to lipid metabolism and endoplasmic reticulum stress markers were analyzed by HPLC, quantitative RT-PCR and western blotting, respectively. Changes in the liver at a histological level were also investigated. The amount of SMP30 mRNA and protein in livers was decreased in Lepr^db/dbSmp30^Y/+ mice compared with Lepr^db/+Smp30^Y/+ mice. Compared with Lepr^db/dbSmp30^Y/+ mice, 24 week old Lepr^db/dbSmp30^Y/− mice showed: i) increased small dense LDL-cho and decreased HDL-cho levels; ii) fatty liver accompanied by numerous inflammatory cells and increased oxidative stress; iii) decreased mRNA expression of genes involved in fatty acid oxidation (PPARα) and lipoprotein uptake (LDLR and VLDLR) but increased CD36 levels; and iv) increased endoplasmic reticulum stress.

Conclusion

Our data strongly suggest that SMP30 is closely associated with NAFLD pathogenesis, and might be a possible therapeutic target for NAFLD.     

Fluorofenidone Offers Improved Renoprotection at Early Interventions during the Course of Diabetic Nephropathy in db/db Mice via Multiple Pathways

Diabetic nephropathy (DN) remains the leading cause of end-stage renal disease (ESRD), a situation that is in part attributable to the lack of effective treatments. Fluorofenidone is a newly developed reagent with anti-fibrotic activity. While fluorofenidone was previously demonstrated to possess renoprotection from DN pathogenesis in db/db mice, the protective process and its underlying mechanisms have not been well studied. To characterize fluorofenidone-derived renoprotection, we treated 5, 8, or 12-week old db/db mice with daily doses of placebo, fluorofenidone, or losartan until 24 weeks of age; the time at which diabetes and DN were fully developed in placebo-treated animals. In comparison to db/db mice receiving fluorofenidone at 12-weeks old, those treated at 5-weeks had less glomerular expansion and better preservation of renal functions, judged by serum creatinine levels, albumin to creatinine ratio, and urinary albumin excretion (mg/24 hours). These benefits of early treatment were associated with significant reductions of multiple DN-promoting events, such as decreased expression of TGF-β1 and the p22^phox subunit of NADPH oxidase as well as downregulated activation of protein kinase C-zeta (ζ), ERK and AKT. This improvement in renoprotection following early interventions is not a unique property of DN pathogenesis, as losartan does not apparently offer the same benefits and is not more renoprotective than fluorofenidone. Additionally, the enhanced renoprotection provided by fluorofenidone did not affect the diabetic process, as it did not alter serum levels of glycated serum proteins, glucose, triglyceride or cholesterol. Collectively, we provide evidence that fluorofenidone offers improved renoprotection at early stages of DN pathogenesis.     

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC beta inhibitor ameliorates not only early diabetes-induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in streptozotocin-induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long-term effects of a PKC beta inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC beta inhibitor reduced urinary albumin excretion rates and inhibited glomerular PKC activation in diabetic db/db mice. Administration of a PKC beta inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF-beta and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long-term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC beta inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.     

Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.     

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 μg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 μg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to N^G-nitro-l-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 μg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOS^Ser1177) and Akt^Ser473, and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state.     

Novel hypoglycemic effects of Ganoderma lucidum water-extract in obese/diabetic (+db/+db) mice

In this study, we evaluated the pharmacological effects of Ganoderma lucidum (G. lucidum) (water-extract) (0.003, 0.03 and 0.3 g/kg, 4-week oral gavage) consumption using the lean (+db/+m) and the obese/diabetic (+db/+db) mice. Different physiological parameters (plasma glucose and insulin levels, lipoproteins-cholesterol levels, phosphoenolpyruvate carboxykinase (PEPCK), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and isolated aorta relaxation of both species were measured and compared. G. lucidum (0.03 and 0.3 g/kg) lowered the serum glucose level in +db/+db mice after the first week of treatment whereas a reduction was observed in +db/+m mice only fed with 0.3 g/kg of G. lucidum at the fourth week. A higher hepatic PEPCK gene expression was found in +db/+db mice. G. lucidum (0.03 and 0.3 g/kg) markedly reduced the PEPCK expression in +db/+db mice whereas the expression of PEPCK was attenuated in +db/+m mice (0.3 g/kg G. lucidum). HMG CoA reductase protein expression (in both hepatic and extra-hepatic organs) and the serum insulin level were not altered by G. lucidum. These data demonstrate that G. lucidum consumption can provide beneficial effects in treating type 2 diabetes mellitus (T2DM) by lowering the serum glucose levels through the suppression of the hepatic PEPCK gene expression.     

Leptin-Dependent Control of Glucose Balance and Locomotor Activity by POMC Neurons

Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways, leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that, specifically, pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility, we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor-deficient Lepr^db/db mice. Here, we show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Lepr^db/db mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Lepr^db/db mice.     

Adiponectin Improves Cardiomyocyte Contractile Function in db/db Diabetic Obese Mice

Low levels of adiponectin, a fat‐derived hormone, are found to be correlated with coronary heart disease, type 2 diabetes, obesity, and insulin resistance. Conversely, high adiponectin levels are predictive of reduced coronary risk in long‐term epidemiologic studies. However, the precise role of adiponectin in cardiomyocyte function is still not clear. This study was designed to examine the role of adiponectin in cardiac contractile function in the db/db model of diabetic obesity. Mechanical properties and intracellular Ca²⁺ transients were evaluated in cardiomyocytes from lean control and db/db mice with or without adiponectin (10 µg/ml) treatment. Expression and phosphorylation of IRS‐1, Akt, c‐Jun, and c‐Jun N terminal kinase (JNK) as well as markers of endoplasmic reticulum (ER) stress were evaluated using western blotting. Cardiomyocytes from db/db mice exhibited greater cross‐sectional area, depressed peak shortening (PS), and maximal velocity of shortening/re‐lengthening as well as prolonged duration of re‐lengthening. Consistently, myocytes from db/db mice displayed a reduced electrically stimulated rise in intracellular Ca²⁺ and prolonged intracellular Ca²⁺ decay, which were abrogated by adiponectin treatment. Ratios between phosphorylated c‐Jun and c‐Jun as well as phosphorylated IRS‐1 and IRS‐1 were increased in db/db mice, the effect of which was attenuated by adiponectin. Levels of the phosphorylated ER stress makers PERK (Thr980), IRE‐1, and eIF2α were significantly elevated in db/db mice compared with lean controls, although the effect was unaffected by adiponectin. Collectively, our data suggest that adiponectin improves cardiomyocyte dysfunction in db/db diabetic obese mice through a mechanism possibly related to c‐Jun and IRS‐1.     

Vascular Endothelial Growth Factor-Receptor 1 Inhibition Aggravates Diabetic Nephropathy through eNOS Signaling Pathway in db/db Mice

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice.While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling.In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.     

An allelic series for the leptin receptor gene generated by CRE and FLP recombinase

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Lepr^db/db mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP/frt and CRE/loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele (Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Lepr^db/db mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Lepr^flox/flox mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in Lepr^17/17 mice, which is indistinguishable from Lepr^neo/neo and Lepr^db/db mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Lepr^neo/neo mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.     

A high-fat diet temporarily renders Sod1-deficient mice resistant to an oxidative insult

Patients with nonalcoholic fatty liver disease may subsequently develop nonalcoholic steatohepatitis after suffering from a second insult, such as oxidative stress. Aim of this study was to investigate the pathogenesis of the liver injury caused when lipids accumulate under conditions of intrinsic oxidative stress using mice that are deficient in superoxide dismutase 1 (SOD1) and the leptin receptor (Lepr). We established Sod1^−/−::Lepr^db/db mice and carried out analyses of four groups of genetically modified mice, namely, wild type, Sod1^−/−, Lepr^db/db and Sod1^−/−::Lepr^db/db mice. Mice with defects in the SOD1 or Lepr gene are vulnerable to developing fatty livers, even when fed a normal diet. Feeding a high-fat diet (HFD) caused an increase in the number of lipid droplets in the liver to different extents in each genotypic mouse. an HFD caused the accelerated death of db/db mice, but contradictory to our expectations, the death rates for the Sod1-deficient mice were decreased by feeding HFD. Consistent with the improved probability of survival, liver damage was significantly ameliorated by feeding an HFD compared to a normal diet in the mice with an Sod1-deficient background. Oxidative stress markers, hyperoxidized peroxiredoxin and lipid peroxidation products, were decreased somewhat in Sod1^−/− mice by feeding HFD. We conclude that lipids reacted with reactive oxygen species and eliminated them in the livers of the young mice, which resulted in the alleviation of oxidative stress, but in advanced age oxidized products accumulated, leading to the aggravation of the liver injury and an increase in fatality rate.     

The db mutation improves memory in younger mice in a model of Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Lepr^db/db) induces mixed or vascular dementia in mature to middle-aged APP^ΔNL/ΔNL x PS1^P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aβ content, which we have not observed at older ages. This was unlikely to be related to altered Aβ synthesis, as both β- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1β mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.     

Renal and glycemic effects of high-dose chromium picolinate in db/db mice: assessment of DNA damage

This study examined renal and glycemic effects of chromium picolinate [Cr(pic)3] supplementation in the context of its purported potential for DNA damage. In preventional protocol, male obese diabetic db/db mice were fed diets either lacking or containing 5, 10 or 100 mg/kg chromium as Cr(pic)3 from 6 to 24 weeks of age; male lean nondiabetic db/m mice served as controls. Untreated db/db mice displayed increased plasma glucose and insulin, hemoglobin A1c, renal tissue advanced glycation end products, albuminuria, glomerular mesangial expansion, urinary 8-hydroxydeoxyguanosine (an index of oxidative DNA damage) and renal tissue immunostaining for γH2AX (a marker of double-strand DNA breaks) compared to db/m controls. Creatinine clearance was lower in untreated db/db mice than their db/m controls, while blood pressure was similar. High Cr(pic)3 intake (i.e., 100-mg/kg diet) mildly improved glycemic status and albuminuria without affecting blood pressure or creatinine clearance. Treatment with Cr(pic)3 did not increase DNA damage despite marked renal accumulation of chromium. In interventional protocol, effects of diets containing 0, 100 and 250 mg/kg supplemental chromium, from 12 to 24 weeks of age, were examined in db/db mice. The results generally revealed similar effects to those of the 100-mg/kg diet of the preventional protocol. In conclusion, the severely hyperglycemic db/db mouse displays renal structural and functional abnormalities in association with DNA damage. High-dose Cr(pic)3 treatment mildly improves glycemic control, and it causes moderate reduction in albuminuria, without affecting the histopathological appearance of the kidney and increasing the risk for DNA damage.     

Cerebral Neovascularization and Remodeling Patterns in Two Different Models of Type 2 Diabetes

We previously reported intense pial cerebral collateralization and arteriogenesis in a mild and lean model of type 2 diabetes (T2D), Goto-Kakizaki (GK) rats. Increased cerebral neovascularization differed regionally and was associated with poor vessel wall maturity. Building upon these findings, the goals of this study were to determine whether a) glycemic control prevents this erratic cerebral neovascularization in the GK model, and b) this pathological neovascularization pattern occurs in Lepr^db/db model, which is the most commonly used model of T2D for studies involving cerebral complications of diabetes. Vascular volume, surface area and structural parameters including microvessel/macrovessel ratio, non-FITC (fluorescein) perfusing vessel abundance, vessel tortuosity, and branch density were measured by 3D reconstruction of FITC stained vasculature in GK rats or Lepr^db/db mice. GK rats exhibited an increase in all of these parameters, which were prevented by glycemic control with metformin. In Lepr^db/db mice, microvascular density was increased but there was no change in nonFITC-perfusing vessels. Increased PA branch density was associated with reduced branch diameter. These results suggest that T2D leads to cerebral neovascularization and remodeling but some structural characteristics of newly formed vessels differ between these models of T2D. The prevention of dysfunctional cerebral neovascularization by early glucose control suggests that hyperglycemia is a mediator of this response.     

Structural,metabolic and endocrine analysis of the diabetes (db/db) hypogonadal syndrome: relationship to hypophyseal hypercytolipidemia

Expression of the diabetes (db/db) mutation in C57BL/KsJ mice results in functional suppression of the female pituitary-gonadal axis accompanied by premature utero-ovarian cytolipoatrophy. Cellular gluco- and lipo-metabolic disturbances promoted by the db/db systemic hyperglycemic-hyperinsulinemic state suppress pituitary gonadotropin release in response to gonadotropin-releasing hormone and gonadal steroid stimulation and results in a hypogonadal-infertility syndrome. Adult female C57BL/KsJ control (+/+ and +/? genotypes) and db/db littermates were monitored for associations in systemic and cellular alterations in luteinizing hormone (LH), follicle-stimulating hormone (FSH), gonadal steroid (binding) levels, and pituitary glucometabolic indices associated with db/db-enhanced lipid imbibition and cytostructural disruption. Obesity, hyperglycemia, and hyperinsulinemia characterized all db/db mutants relative to controls. Serum and pituitary progesterone and estradiol concentrations were suppressed in db/db mutants, in association with serum LH and FSH levels, but not with pituitary LH and FSH concentrations, which were comparable between groups. Pituitary insulin receptor binding and glucose utilization rates were suppressed in db/db groups relative to +/? indices. Structural and cytochemical analysis of anterior (AP), intermediate (IL), and neuro-(NP) hypophyseal lobes demonstrated prominent hypercytolipidemia in db/db mutants relative to controls. Prominent cytolipidemia was localized within well-granulated basophilic gonadotrophs and within IL and NP pituicytes. Vasolipidemia and interstitial cytoadiposity were prominent throughout all db/db pituitary lobes. Thus, disturbances associated with pituitary hypercytolipidemia are functional components of the expressed diabetes-associated hypogonadal syndrome in db/db mutants. Progressive alterations in hypophyseal cytoarchitecture are correlated with suppression of pituitary metabolic and endocrine indices, alterations that contribute to functional disruption of the pituitary-hypogonadal axis in C57BL/KsJ-db/db mice.

     

Chlorogenic Acid Improves Late Diabetes through Adiponectin Receptor Signaling Pathways in db/db Mice

The aim of this study was to examine the effects of chlorogenic acid (CGA) on glucose and lipid metabolism in late diabetic db/db mice, as well as on adiponectin receptors and their signaling molecules, to provide evidence for CGA in the prevention of type 2 diabetes. We randomly divided 16 female db/db mice into db/db-CGA and db/db-control (CON) groups equally; db/m mice were used as control mice. The mice in both the db/db-CGA and db/m-CGA groups were administered 80 mg/kg/d CGA by lavage for 12 weeks, whereas the mice in both CON groups were given equal volumes of phosphate-buffered saline (PBS) by lavage. At the end of the intervention, we assessed body fat and the parameters of glucose and lipid metabolism in the plasma, liver and skeletal muscle tissues as well as the levels of aldose reductase (AR) and transforming growth factor-β1 (TGF-β1) in the kidneys and measured adiponectin receptors and the protein expression of their signaling molecules in liver and muscle tissues. After 12 weeks of intervention, compared with the db/db-CON group, the percentage of body fat, fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c) in the db/db-CGA group were all significantly decreased; TGF-β1 protein expression and AR activity in the kidney were both decreased; and the adiponectin level in visceral adipose was increased. The protein expression of adiponectin receptors (ADPNRs), the phosphorylation of AMP-activated protein kinase (AMPK) in the liver and muscle, and the mRNA and protein levels of peroxisome proliferator-activated receptor alpha (PPAR-α) in the liver were all significantly greater. CGA could lower the levels of fasting plasma glucose and HbA1c during late diabetes and improve kidney fibrosis to some extent through the modulation of adiponectin receptor signaling pathways in db/db mice.     

Bile acid sequestration reduces plasma glucose levels in db/db mice by increasing its metabolic clearance rate

Aims/Hypothesis

Bile acid sequestrants (BAS) reduce plasma glucose levels in type II diabetics and in murine models of diabetes but the mechanism herein is unknown. We hypothesized that sequestrant-induced changes in hepatic glucose metabolism would underlie reduced plasma glucose levels. Therefore, in vivo glucose metabolism was assessed in db/db mice on and off BAS using tracer methodology.

Methods

Lean and diabetic db/db mice were treated with 2% (wt/wt in diet) Colesevelam HCl (BAS) for 2 weeks. Parameters of in vivo glucose metabolism were assessed by infusing [U-¹³C]-glucose, [2-¹³C]-glycerol, [1-²H]-galactose and paracetamol for 6 hours, followed by mass isotopologue distribution analysis, and related to metabolic parameters as well as gene expression patterns.

Results

Compared to lean mice, db/db mice displayed an almost 3-fold lower metabolic clearance rate of glucose (p = 0.0001), a ∼300% increased glucokinase flux (p = 0.001) and a ∼200% increased total hepatic glucose production rate (p = 0.0002). BAS treatment increased glucose metabolic clearance rate by ∼37% but had no effects on glucokinase flux nor total hepatic or endogenous glucose production. Strikingly, BAS-treated db/db mice displayed reduced long-chain acylcarnitine content in skeletal muscle (p = 0.0317) but not in liver (p = 0.189). Unexpectedly, BAS treatment increased hepatic FGF21 mRNA expression 2-fold in lean mice (p = 0.030) and 3-fold in db/db mice (p = 0.002).

Conclusions/Interpretation

BAS induced plasma glucose lowering in db/db mice by increasing metabolic clearance rate of glucose in peripheral tissues, which coincided with decreased skeletal muscle long-chain acylcarnitine content.     

Gene Expression of Inflammatory Cytokines in Peripheral Leukocytes in db/db Mice Rose with Progression of Diabetes

Fourteen-week-old db/db mice showed significant higher blood glucose levels than 7 week-old mice. The mRNA levels of IL-1β and S100a4/a6/a9 in peripheral leukocytes were higher in 14 week-old mice than in 7 week-old mice. Together, inflammatory cytokine/cytokine-like factor mRNA levels in peripheral leukocytes were associated with progression of diabetes in db/db mice.     

Oxamate Improves Glycemic Control and Insulin Sensitivity via Inhibition of Tissue Lactate Production in db/db Mice

Oxamate (OXA) is a pyruvate analogue that directly inhibits the lactate dehydrogenase (LDH)-catalyzed conversion process of pyruvate into lactate. Earlier and recent studies have shown elevated blood lactate levels among insulin-resistant and type 2 diabetes subjects and that blood lactate levels independently predicted the development of incident diabetes. To explore the potential of OXA in the treatment of diabetes, db/db mice were treated with OXA in vivo. Treatment of OXA (350–750 mg/kg of body weight) for 12 weeks was shown to decrease body weight gain and blood glucose and HbA1c levels and improve insulin secretion, the morphology of pancreatic islets, and insulin sensitivity in db/db mice. Meanwhile, OXA reduced the lactate production of adipose tissue and skeletal muscle and serum lactate levels and decreased serum levels of TG, FFA, CRP, IL-6, and TNF-α in db/db mice. The PCR array showed that OXA downregulated the expression of Tnf, Il6, leptin, Cxcr3, Map2k1, and Ikbkb, and upregulated the expression of Irs2, Nfkbia, and Pde3b in the skeletal muscle of db/db mice. Interestingly, LDH-A expression increased in the islet cells of db/db mice, and both treatment of OXA and pioglitazone decreased LDH-A expression, which might be related to the improvement of insulin secretion. Taken together, increased lactate production of adipose tissue and skeletal muscle may be at least partially responsible for insulin resistance and diabetes in db/db mice. OXA improved glycemic control and insulin sensitivity in db/db mice primarily via inhibition of tissue lactate production. Oxamic acid derivatives may be a potential drug for the treatment of type 2 diabetes.     

Therapeutic Effects of Fenofibrate on Diabetic Peripheral Neuropathy by Improving Endothelial and Neural Survival in db/db Mice

Neural vascular insufficiency plays an important role in diabetic peripheral neuropathy (DPN). Peroxisome proliferative-activated receptor (PPAR)α has an endothelial protective effect related to activation of PPARγ coactivator (PGC)-1α and vascular endothelial growth factor (VEGF), but its role in DPN is unknown. We investigated whether fenofibrate would improve DPN associated with endothelial survival through AMPK-PGC-1α-eNOS pathway. Fenofibrate was given to db/db mice in combination with anti-flt-1 hexamer and anti-flk-1 heptamer (VEGFR inhibition) for 12 weeks. The db/db mice displayed sensory-motor impairment, nerve fibrosis and inflammation, increased apoptotic cells, disorganized myelin with axonal shrinkage and degeneration, fewer unmyelinated fibers, and endoneural vascular rarefaction in the sciatic nerve compared to db/m mice. These findings were exacerbated with VEGFR inhibition in db/db mice. Increased apoptotic cell death and endothelial dysfunction via inactivation of the PPARα-AMPK-PGC-1α pathway and their downstream PI3K-Akt-eNOS-NO pathway were noted in db/db mice, human umbilical vein endothelial cells (HUVECs) and human Schwann cells (HSCs) in high-glucose media. The effects were more prominent in response to VEGFR inhibition. In contrast, fenofibrate treatment ameliorated neural and endothelial damage by activating the PPARα-AMPK-PGC-1α-eNOS pathway in db/db mice, HUVECs and HSCs. Fenofibrate could be a promising therapy to prevent DPN by protecting endothelial cells through VEGF-independent activation of the PPARα-AMPK-PGC-1α-eNOS-NO pathway.