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.     

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.     

Morphological and functional adaptation of pancreatic islet blood vessels to insulin resistance is impaired in diabetic db/db mice

The pancreatic islet vasculature is of fundamental importance to the β-cell response to obesity-associated insulin resistance. To explore islet vascular alterations in the pathogenesis of type 2 diabetes, we evaluated two insulin resistance models: ob/ob mice, which sustain large β-cell mass and hyperinsulinemia, and db/db mice, which progress to diabetes due to secondary β-cell compensation failure for insulin secretion. Time-dependent changes in islet vasculature and blood flow were investigated using tomato lectin staining and in vivo live imaging. Marked islet capillary dilation was observed in ob/ob mice, but this adaptive change was blunted in db/db mice. Islet blood flow volume was augmented in ob/ob mice, whereas it was reduced in db/db mice. The protein concentrations of total and phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 were increased in ob/ob islets, while they were diminished in db/db mice, indicating decreased eNOS activity. This was accompanied by an increased retention of advanced glycation end-products in db/db blood vessels. Amelioration of diabetes by Elovl6 deficiency involved a restoration of capillary dilation, blood flow, and eNOS phosphorylation in db/db islets. Our findings suggest that the disability of islet capillary dilation due to endothelial dysfunction impairs local islet blood flow, which may play a role in the loss of β-cell function and further exacerbate type 2 diabetes.     

In vivo evidence of enhanced di-methylation of histone H3 K4 on upregulated genes in adipose tissue of diabetic db/db mice

Di-methylation of histone H3 lysine (K) 4, a component of the epigenetic memory, is associated with gene transactivation. In this study, we examined whether the development of diabetes induces di-methylation of histone H3 K4 on the upregulated genes. We searched for upregulated genes in mesenteric adipose tissue of insulin-resistant/diabetic db/db mice compared with non-diabetic db/m mice using microarray analysis. We also performed chromatin immunoprecipitation assays for di-methylation of histone H3 K4 in the upregulated genes in mesenteric adipose tissue of db/m and db/db mice. Di-methylation of histone H3 K4 was enhanced at the upstream and/or transcribed regions of upregulated genes including Atp6v0d2, Mmp12, Trem2 and Clec4d genes in mesenteric adipose tissue of db/db mice, as compared with db/m mice. These results suggest that di-methylation of histone H3 K4 is involved in the induction of Atp6v0d2, Mmp12, Trem2 and Clec4d in mesenteric adipose tissue in db/db mice.     

Constitutive hepatic glucokinase activity db/db and ob/ob mice

The specific activity of hepatic glucokinase (ATP: d-glucose 6-phosphotransferase, EC 2.7.1.2) in db/db mice and ob/ob mice was higher than in normal mice. All enzymes had a similar K_m and, thus, the difference in activity was not due to differences in the affinity of enzyme molecules to substrates. Mixing liver extracts with high or low enzyme activities yielded additive results, as expected, which ruled out the involvement of an inhibitor or activator of the enzyme. Fasting normal mice of either strain for three days decreased glucokinase activity. However, fasting db/db or ob/ob mice for as long as 10 days had no effect on enzyme activity, indicating that glucokinase in db/db or ob/ob mice was out of regulation of constitutive. The constant, abnormally high glucokinase activity may be a contributing factor to the obesity of ob/ob or db/db mice. These mice provide a model system to study the regulation of this rate-limiting enzyme of glucose metabolism.     

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice

Background

Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice.

Results

Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice.

Conclusions

These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.     

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.     

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.     

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.

     

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.     

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.     

Chronic stress aggravates glucose intolerance in leptin receptor-deficient (db/db) mice

Genetic predisposition and environmental challenges interact to determine individual vulnerability to obesity and type 2 diabetes. We previously established a mouse model of chronic subordination stress-induced hyperphagia, obesity, metabolic like-syndrome and insulin resistance in the presence of a high-fat diet. However, it remains to be established if social stress could also aggravate glucose intolerance in subjects genetically predisposed to develop obesity and type 2 diabetes. To answer this question, we subjected genetically obese mice due to deficiency of the leptin receptor (db/db strain) to chronic subordination stress. Over five weeks, subordination stress in db/db mice led to persistent hyperphagia, hyperglycemia and exacerbated glucose intolerance altogether suggestive of an aggravated disorder when compared to controls. On the contrary, body weight and fat mass were similarly affected in stressed and control mice likely due to the hyperactivity shown by subordinate mice. Stressed db/db mice also showed increased plasma inflammatory markers. Altogether our results suggest that chronic stress can aggravate glucose intolerance but not obesity in genetically predisposed subjects on the basis of a disrupted leptin circuitry.     

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.     

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.     

Jejunal Proteins Secreted by db/db Mice or Insulin-Resistant Humans Impair the Insulin Signaling and Determine Insulin Resistance

Background

Two recent studies demonstrated that bariatric surgery induced remission of type 2 diabetes very soon after surgery and far too early to be attributed to weight loss. In this study, we sought to explore the mechanism/s of this phenomenon by testing the effects of proteins from the duodenum-jejunum conditioned-medium (CM) of db/db or Swiss mice on glucose uptake in vivo in Swiss mice and in vitro in both Swiss mice soleus and L6 cells. We studied the effect of sera and CM proteins from insulin resistant (IR) and insulin-sensitive subjects on insulin signaling in human myoblasts.

Methodology/Principal Findings

db/db proteins induced massive IR either in vivo or in vitro, while Swiss proteins did not. In L6 cells, only db/db proteins produced a noticeable increase in basal ⁴⁷³Ser-Akt phosphorylation, lack of GSK3β inhibition and a reduced basal ³⁸⁹Thr-p70-S6K1 phosphorylation. Human IR serum markedly increased basal ⁴⁷³Ser-Akt phosphorylation in a dose-dependent manner. Human CM IR proteins increased by about twofold both basal and insulin-stimulated ⁴⁷³Ser-Akt. Basal ⁹Ser-GSK3β phosphorylation was increased by IR subjects serum with a smaller potentiating effect of insulin.

Conclusions

These findings show that jejunal proteins either from db/db mice or from insulin resistant subjects impair muscle insulin signaling, thus inducing insulin resistance.     

Responses of the Root Systems of Sunflower and Maize to Unidirectional Stem Flexure

Plants of two contrasting species of herbaceous annuals, thedicot sunflower (Helianthus annuusL.) and the monocot maize(Zea maysL.), grown in the glasshouse were subjected to regularunidirectional stem flexure. Differences in morphology and mechanicalproperties of roots and shoots were then investigated. Rootsystems were divided into quadrants around the axis of stimulationand differences in root morphology and mechanics between thezones were investigated. There were considerable differencesbetween roots in the leeward and windward zones compared withroots perpendicular to the axis of stimulation. First-orderlateral roots in both species were thicker, more rigid and morenumerous. These results suggest that plant roots respond locallyto mechanical stimulation. There were, however, also differencesin the responses of the two species. In sunflower, the tap rootand stem base became elliptical in cross section with the majoraxis lying in the plane of stimulation. The lateral roots offlexed sunflowers in both the leeward and windward zones showedsimilar growth responses: roots were thicker, more numerousand weighed more than those in the perpendicular zones. However,only leeward roots showed significant differences in their mechanicalproperties; roots were more rigid, stronger and stiffer. Incontrast, the leeward roots of maize were thicker and more numerous,with a greater biomass than the windward roots. However, onlyroots in the windward zone were stiffer than those in the perpendicularzone. These differences between sunflower and maize are relatedto their contrasting anchorage mechanics.Copyright 1998 Annalsof Botany Company Anchorage, biomechanics, adaptive growth, roots, thigmomorphogenesis,Helianthus annuusL.,Zea maysL.     

Long-Term Supranutritional Supplementation with Selenate Decreases Hyperglycemia and Promotes Fatty Liver Degeneration by Inducing Hyperinsulinemia in Diabetic db/db Mice

There are conflicting reports on the link between the micronutrient selenium and the prevalence of diabetes. To investigate the possibility that selenium acts as a “double-edged sword” in diabetes, cDNA microarray profiling and two-dimensional differential gel electrophoresis coupled with mass spectrometry were used to determine changes in mRNA and protein expression in pancreatic and liver tissues of diabetic db/db mice in response to dietary selenate supplementation. Fasting blood glucose levels increased continuously in db/db mice administered placebo (DMCtrl), but decreased gradually in selenate-supplemented db/db mice (DMSe) and approached normal levels after termination of the experiment. Pancreatic islet size was increased in DMSe mice compared with DMCtrl mice, resulting in a clear increase in insulin production and a doubling of plasma insulin concentration. Genes that encode proteins involved in key pancreatic β-cell functions, including regulation of β-cell proliferation and differentiation and insulin synthesis, were found to be specifically upregulated in DMSe mice. In contrast, apoptosis-associated genes were downregulated, indicating that islet function was protected by selenate treatment. Conversely, liver fat accumulation increased in DMSe mice together with significant upregulation of lipogenic and inflammatory genes. Genes related to detoxification were downregulated and antioxidant enzymatic activity was reduced, indicating an unexpected reduction in antioxidant defense capacity and exacerbation of fatty liver degeneration. Moreover, proteomic analysis of the liver showed differential expression of proteins involved in glucolipid metabolism and the endoplasmic reticulum assembly pathway. Taken together, these results suggest that dietary selenate supplementation in db/db mice decreased hyperglycemia by increasing insulin production and secretion; however, long-term hyperinsulinemia eventually led to reduced antioxidant defense capacity, which exacerbated fatty liver degeneration.     

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.     

Dietary flavonoids suppress azoxymethane-induced colonic preneoplastic lesions in male C57BL/KsJ-db/db mice

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68–91% and BCAC by 64–71%, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans.     

Sex-limited effects of the expression of the db gene in mice during puberty

The autosomal recessive gene diabetes (db) produces a condition similar to human insulin-dependent diabetes mellitus in certain strains of inbred mice. In this investigation, the effects of expression of the db gene on the development of the submandibular glands, electrophoretic protein patterns in salivas, fasting blood glucose levels, and glycosylated hemoglobin levels were evaluated in mice undergoing puberty. Three sex-limited effects of the db gene were observed in diabetic male mice: (1) a compromise of the development of the specialized submandibular glands with the extensive tubular portion normally found in males, (2) failure to develop a salivary protein pattern unique to male mice, and (3) attainment of higher levels of fasting blood glucose than found in female diabetic mice. Since it has been documented that homozygous mice fail to develop functional gonads, apparently due to insufficient production of gonadotropin, it is likely that the compromised development of the specialized submandibular glands, and, consequently, the male salivary protein pattern, is a result of decreased testosterone production. Experiments in which diabetic mice were treated with testosterone support that conclusion, since testosterone caused transformation of the salivary protein pattern to one identical with that of normal male littermate controls and increased the tubular portion of the submandibular glands.This study was supported in part by Public Health Service Research Grant 5 R01 AM21177 and by the Indiana University Human Genetics Center (PHS P01 GM21054). The author was supported by Public Health Service Career Development Award 1 K04 AM00284. This is Publication No. 79-18 from the Indiana University Human Genetics Center.