Analysis of gene expression profiles in insulin-sensitive tissues from pre-diabetic and diabetic Zucker diabetic fatty rats

Insulin resistance occurs early in the disease process, preceding the development of type 2 diabetes. Therefore, the identification of molecules that contribute to insulin resistance and leading up to type 2 diabetes is important to elucidate the molecular pathogenesis of the disease. To this end, we characterized gene expression profiles from insulin-sensitive tissues, including adipose tissue, skeletal muscle, and liver tissue of Zucker diabetic fatty (ZDF) rats, a well characterized type 2 diabetes animal model. Gene expression profiles from ZDF rats at 6 weeks (pre-diabetes), 12 weeks (diabetes), and 20 weeks (late-stage diabetes) were compared with age- and sex-matched Zucker lean control (ZLC) rats using 5000 cDNA chips. Differentially regulated genes demonstrating > 1.3-fold change at age were identified and categorized through hierarchical clustering analysis. Our results showed that while expression of lipolytic genes was elevated in adipose tissue of diabetic ZDF rats at 12 weeks of age, expression of lipogenic genes was decreased in liver but increased in skeletal muscle of 12 week old diabetic ZDF rats. These results suggest that impairment of hepatic lipogenesis accompanied with the reduced lipogenesis of adipose tissue may contribute to development of diabetes in ZDF rats by increasing lipogenesis in skeletal muscle. Moreover, expression of antioxidant defense genes was decreased in the liver of 12-week old diabetic ZDF rats as well as in the adipose tissue of ZDF rats both at 6 and 12 weeks of age. Cytochrome P450 (CYP) genes were also significantly reduced in 12 week old diabetic liver of ZDF rats. Genes involved in glucose utilization were downregulated in skeletal muscle of diabetic ZDF rats, and the hepatic gluconeogenic gene was upregulated in diabetic ZDF rats. Genes commonly expressed in all three tissue types were also observed. These profilings might provide better fundamental understanding of insulin resistance and development of type 2 diabetes.     

Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma

BACKGROUND/AIMS: It has been known for at least 50 years that alterations in methionine metabolism occur in human liver cirrhosis. However, the molecular basis of this alteration is not completely understood. In order to gain more insight into the mechanisms behind this condition, mRNA levels of methionine adenosyltransferase (MAT1A), glycine methyltransferase (GNMT), methionine synthase (MS), betaine homocysteine methyltransferase (BHMT) and cystathionine beta-synthase (CBS) were examined in 26 cirrhotic livers, five hepatocellular carcinoma (HCC) tissues and ten control livers. METHODS: The expression of the above-mentioned genes was determined by quantitative RT-PCR analysis. Methylation of MAT1A promoter was assessed by methylation-sensitive restriction enzyme digestion of genomic DNA. RESULTS: When compared to normal livers MAT1A, GNMT, BHMT, CBS and MS mRNA contents were significantly reduced in liver cirrhosis. Interestingly, MAT1A promoter was hypermethylated in the cirrhotic liver. HCC tissues also showed decreased mRNA levels of these enzymes. CONCLUSIONS: These findings establish that the abundance of the mRNA of the main genes involved in methionine metabolism is markedly reduced in human cirrhosis and HCC. Hypermethylation of MAT1A promoter could participate in its reduced expression in cirrhosis. These observations help to explain the hypermethioninemia, hyperhomocysteinemia and reduced hepatic glutathione content observed in cirrhosis.     

Melatonin reduces hepatic mitochondrial dysfunction in diabetic obese rats

Hepatic mitochondrial dysfunction is thought to play a role in the development of liver steatosis and insulin resistance, which are both common characteristics of obesity and type 2 diabetes mellitus (T2DM). It was hypothesized that the antioxidant properties of melatonin could potentially improve the impaired functions of hepatic mitochondria in diabetic obese animals. Male Zucker diabetic fatty (ZDF) rats and lean littermates (ZL) were given either melatonin (10 mg/kg BW/day) orally for 6 wk (M‐ZDF and M‐ZL) or vehicle as control groups (C‐ZDF and C‐ZL). Hepatic function was evaluated by measurement of serum alanine transaminase and aspartate transaminase levels, liver histopathology and electron microscopy, and hepatic mitochondrial functions. Several impaired functions of hepatic mitochondria were observed in C‐ZDF in comparison with C‐ZL rats. Melatonin treatment to ZDF rats decreases serum levels of ALT (P < 0.001), alleviates liver steatosis and vacuolation, and also mitigates diabetic‐induced mitochondrial abnormalities, glycogen, and lipid accumulation. Melatonin improves mitochondrial dysfunction in M‐ZDF rats by increasing activities of mitochondrial citrate synthase (P < 0.001) and complex IV of electron transfer chain (P < 0.05) and enhances state 3 respiration (P < 0.001), respiratory control index (RCR) (P < 0.01), and phosphorylation coefficient (ADP/O ratio) (P < 0.05). Also melatonin augments ATP production (P < 0.05) and diminishes uncoupling protein 2 levels (P < 0.001). These results demonstrate that chronic oral melatonin reduces liver steatosis and mitochondria dysfunction in ZDF rats. Therefore, it may be beneficial in the treatment of diabesity.     

Adiponectin receptors: expression in Zucker diabetic rats and effects of fenofibrate and metformin

The insulin-sensitizing adipokine, adiponectin, acts through 2 receptors, AdipoR1 and AdipoR2. A decreased expression of these receptors could contribute to insulin resistance and diabetes. We determined if the expression of adiponectin receptors is decreased in an experimental model, the Zucker diabetic rat (ZDF), and if a peroxisome proliferator-activated receptor alpha agonist, fenofibrate, and metformin could increase these expressions. The ZDF and control (L) rats were studied at 7, 14, and 21 weeks. After initial study at 7 weeks, ZDF received no treatment (n = 10), metformin (n = 10), or fenofibrate (n = 10) until final studies at 14 or 21 weeks. The L rats received no treatment. AdipoR1 and R2 expressions were measured in liver, muscle, and white adipose tissue (WAT). As expected, ZDF rats were insulin resistant at 7 weeks, had type 2 diabetes mellitus at 14 weeks, and had diabetes with insulin deficiency at 21 weeks. Compared with L rats, AdipoRs messenger RNA was decreased only in the WAT (P < .05) of 7-week-old ZDF rats, but was unchanged in muscle and increased in liver. Metformin and fenofibrate decreased plasma triacylglycerols (P < .01) as expected. The only effect of fenofibrate on AdipoRs was a moderate increase (P < .01) of both receptors' messenger RNA in liver. Metformin increased AdipoR1 and R2 expression in muscle (P < .01) and AdipoR1 (P < .01) in WAT. These results do not support an important role for decreased AdipoRs expression in the development of insulin resistance and diabetes. Parts of the actions of fenofibrate and of metformin could be mediated by a stimulation of the expression of these receptors in liver and in insulin-sensitive, glucose-utilizing tissues (muscle, WAT), respectively.     

Treatment of Zucker diabetic fatty rats with AVE7688 improves vascular and neural dysfunction

Aim:  Vasopeptidase inhibitors are drugs that inhibit angiotensin-converting enzyme and neutral endopeptidase (NEP). The latter is a protease that degrades vasoactive peptides and is increased in diabetes. We have previously shown that treating streptozotocin-induced diabetic rats, an animal model of type 1 diabetes, with AVE7688, a vasopeptidase inhibitor, improves neurovascular and neural function. In this study, we determined the effect of treating Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes, with AVE7688 on vascular and neural function.
Methods:  ZDF rats at 12 weeks of age were treated for 12 weeks with AVE7688 (500 mg/kg diet). Afterwards, vascular reactivity of epineurial arterioles of the sciatic nerve and nerve conduction velocity and blood flow was determined.
Results:  Vascular and neural function was significantly impaired in ZDF rats compared with age-matched lean (control) rats. Treating ZDF rats with AVE7688 improved vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineurial arterioles. Motor and sensory nerve conduction velocity, endoneurial blood flow and thermal nociception end-points were also improved by treatment compared with untreated ZDF rats. Superoxide and expression of NEP were increased in epineurial arterioles from ZDF rats and attenuated by treatment with AVE7688.
Conclusions:  AVE7688 is an effective treatment for microvascular and neural disease in ZDF rats. Thus, vasopeptidase inhibitors may be an effective treatment for diabetic microvascular and neural complication in type 2 diabetes.     

Taspoglutide, a novel human once-weekly GLP-1 analogue, protects pancreatic β-cells in vitro and preserves islet structure and function in the Zucker diabetic fatty rat in vivo

Aim: Glucagon‐like peptide‐1 (GLP‐1) has protective effects on pancreatic β‐cells. We evaluated the effects of a novel, long‐acting human GLP‐1 analogue, taspoglutide, on β‐cells in vitro and in vivo. Methods: Proliferation of murine pancreatic β (MIN6B1) cells and rat islets in culture was assessed by imaging of 5‐ethynyl‐2′‐deoxyuridine‐positive cells after culture with taspoglutide. Apoptosis was evaluated with the transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate nick‐end labelling assay in rat insulinoma (INS‐1E) cells and isolated human islets exposed to cytokines (recombinant interleukin‐1β, interferon‐γ, tumour necrosis factor‐α) or lipotoxicity (palmitate) in the presence or absence of taspoglutide. Islet morphology and survival and glucose‐stimulated insulin secretion in perfused pancreata were assessed 3–4 weeks after a single application of taspoglutide to prediabetic 6‐week‐old male Zucker diabetic fatty (ZDF) rats. Results: Proliferation was increased in a concentration‐dependent manner up to fourfold by taspoglutide in MIN6B1 cells and was significantly stimulated in isolated rat islets. Taspoglutide almost completely prevented cytokine‐ or lipotoxicity‐induced apoptosis in INS‐1E cells (control 0.5%, cytokines alone 2.2%, taspoglutide + cytokines 0.6%, p < 0.001; palmitate alone 8.1%, taspoglutide + palmitate 0.5%, p < 0.001) and reduced apoptosis in isolated human islets. Treatment of ZDF rats with taspoglutide significantly prevented β‐cell apoptosis and preserved healthy islet architecture and insulin staining intensity as shown in pancreatic islet cross sections. Basal and glucose‐stimulated insulin secretion of in situ perfused ZDF rat pancreata was normalized after taspoglutide treatment. Conclusions: Taspoglutide promoted β‐cell proliferation, prevented apoptosis in vitro and exerted multiple β‐cell protective effects on islet architecture and function in vivo in ZDF rats.     

Salacia oblonga root decreases cardiac hypertrophy in Zucker diabetic fatty rats: inhibition of cardiac expression of angiotensin II type 1 receptor

Aims: We investigated the effect of the water extract of Salacia oblonga (SOE), an ayurvedic antidiabetic and antiobesity medicine, on obesity and diabetes‐associated cardiac hypertrophy and discuss the role of modulation of cardiac angiotensin II type 1 receptor (AT₁) expression in the effect. Methods: SOE (100 mg/kg) was given orally to male Zucker diabetic fatty (ZDF) rats for 7 weeks. At the end‐point of the treatment, the hearts and left ventricles were weighed, cardiomyocyte cross‐sectional areas were measured, and cardiac gene profiles were analysed. On the other hand, angiotensin II–stimulated embryonic rat heart–derived H9c2 cells and neonatal rat cardiac fibroblasts were pretreated with SOE and one of its prominent components mangiferin (MA), respectively. Atrial natriuretic peptide (ANP) mRNA expression and protein synthesis and [³H]thymidine incorporation were determined. Results: SOE‐treated ZDF rats showed less cardiac hypertrophy (decrease in weights of the hearts and left ventricles and reduced cardiomyocyte cross‐sectional areas). SOE treatment suppressed cardiac overexpression of ANP, brain natriuretic peptide (BNP) and AT₁ mRNAs and AT₁ protein in ZDF rats. SOE (50–100 μg/ml) and MA (25 μmol) suppressed angiotensin II–induced ANP mRNA overexpression and protein synthesis in H9c2 cells. They also inhibited angiotensin II–stimulated [³H]thymidine incorporation by cardiac fibroblasts. Conclusions: Our findings demonstrate that SOE decreases cardiac hypertrophy in ZDF rats, at least in part by inhibiting cardiac AT₁ overexpression. These studies provide insights into a potential cardioprotective role of a traditional herb, which supports further clinical evaluation in obesity and diabetes‐associated cardiac hypertrophy.     

JTT-130, a novel intestine-specific inhibitor of microsomal triglyceride transfer protein, ameliorates impaired glucose and lipid metabolism in Zucker diabetic fatty rats

Aim: Microsomal triglyceride transfer protein (MTP) takes part in the mobilization of triglyceride‐rich lipoproteins from enterocytes and hepatocytes. We investigated the effects of JTT‐130, a novel intestine‐specific MTP inhibitor, on impaired glucose and lipid metabolism in Zucker diabetic fatty (ZDF) rats. Methods: Male ZDF rats were fed a regular powdered diet with or without JTT‐130 as a food admixture (0.01–0.02%) for 6 weeks. Food intake, body weight, blood biochemical parameters, fecal lipid contents, hepatic lipid contents, tissue mRNA levels and glucose utilization in adipose tissues were assessed. An intraperitoneal glucose tolerance test (IPGTT) and histological analysis of the pancreas were performed. Results: JTT‐130 treatment decreased food intake, glycated hemoglobin, plasma levels of glucose, triglycerides and total cholesterol, hepatic levels of triglycerides and cholesterol and hepatic mRNA levels of glucose‐6‐phosphatase, phosphoenolpyruvate carboxykinase and fructose‐1,6‐bisphosphatase. JTT‐130 treatment increased fecal levels of free fatty acids and cholesterol, plasma levels of glucagon‐like peptide‐1 and peptide YY, mRNA levels of glucose transporter 4 (GLUT4) and lipoprotein lipase in adipose tissues and GLUT4 in muscle and glucose utilization in adipose tissues. Plasma insulin decreased after 2 weeks and increased after 4 weeks of JTT‐130 treatment. Plasma glucose in the JTT‐130‐treated rats was lower with higher plasma insulin than in the control rats during the IPGTT. The islets of the JTT‐130‐treated rats were larger and contained more insulin than those of the control rats. Conclusions: JTT‐130 ameliorates impaired glucose and lipid metabolism in the ZDF rats thereby suggesting that JTT‐130 could be useful for prevention and treatment of type 2 diabetes.     

Antidiabetic and antihypertensive effect of a polyphenol-rich fraction of Thymelaea hirsuta L. in a model of neonatal streptozotocin-diabetic and N(G) -nitro-l-arginine methyl ester-hypertensive rats

Background: The present study examined the effect of the polyphenol‐rich fraction from Thymelaea hirsuta (PRF‐Th) in a rat model of streptozotocin (STZ)‐diabetes and nitric oxide (NO)‐deficient hypertension. Methods: Diabetes was induced by a single dose of STZ (90 mg/kg, i.p.). To induce NO‐deficient hypertension, rats were treated with the NO synthase inhibitor N^G‐nitro‐l ‐arginine methyl ester; l ‐NAME; 30 mg/kg per day, p.o. for 3 weeks. The effects of 21 days treatment with 80 mg/kg per day PRF‐Th in the drinking water were evaluated in diabetic–hypertensive (DH) rats. In all groups (n = 6 in each), glycemia and systolic blood pressure were determined weekly. At the end of the experiment, hepatic glycogen was determined. Results: Blood glucose levels decreased gradually from baseline until the end of the experiment in untreated DH rats (from 1.92 ± 0.09 to 1.3 ± 0.1 g/L; P < 0.05). Administration of PRF‐Th concomitantly with l ‐NAME prevented the blood pressure increase in rats. After 21 days, blood pressure in PRF‐Th + l ‐NAME‐ and l ‐NAME‐treated rats was 132 ± 1 and 157 ± 1 mmHg, respectively (P < 0.001). Administration of 2 mL/kg per day PRF‐Th to DH rats significantly increased hepatic glycogen levels compared with levels in untreated DH rats (13.65 ± 1.84 vs 6.34 ± 0.75 mg/g tissue, respectively; P < 0.01). Moreover, PRF‐Th significantly reduced the amount of glucose absorbed in in situ perfused jejunum segments compared with control (by 33.6%; P < 0.001). This effect of PRF‐Th was comparable with that of acarbose, an α‐glucosidase inhibitor. Conclusions: The findings of the present study indicate that T. hirsuta has antidiabetic and antihypertensive activity in STZ‐diabetic, NO‐deficient hypertensive rats. This effect seems to be due to its rich polyphenol content. Therefore, T. hirsuta may be useful as a food supplement for the prevention of type 2 diabetes and hypertension.     

Melatonin induces browning of inguinal white adipose tissue in Zucker diabetic fatty rats

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Identification of brown fat (beige/brite) in white adipose tissue (WAT) prompted us to investigate whether melatonin is a brown‐fat inducer. We used Zücker diabetic fatty (ZDF) rats, a model of obesity‐related type 2 diabetes and a strain in which melatonin reduces obesity and improves their metabolic profiles. At 5 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control and those treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk. Melatonin induced browning of inguinal WAT in both ZDF and ZL rats. Hematoxylin–eosin staining showed patches of brown‐like adipocytes in inguinal WAT in ZDF rats and also increased the amounts in ZL animals. Inguinal skin temperature was similar in untreated lean and obese rats. Melatonin increased inguinal temperature by 1.36 ± 0.02°C in ZL and by 0.55 ± 0.04°C in ZDF rats and sensitized the thermogenic effect of acute cold exposure in both groups. Melatonin increased the amounts of thermogenic proteins, uncoupling protein 1 (UCP1) (by ~2‐fold, P < 0.01) and PGC‐1α (by 25%, P < 0.05) in extracts from beige inguinal areas in ZL rats. Melatonin also induced measurable amounts of UCP1 and stimulated by ~2‐fold the levels of PGC‐1α in ZDF animals. Locomotor activity and circulating irisin levels were not affected by melatonin. These results demonstrate that chronic oral melatonin drives WAT into a brown‐fat‐like function in ZDF rats. This may contribute to melatonin′s control of body weight and its metabolic benefits.     

Vasopeptidase inhibition prevents nephropathy in Zucker diabetic fatty rats

BACKGROUND: Blocking the renin-angiotensin system is an established therapeutic principle in diabetic nephropathy. We investigated whether inhibition of both neutral endopeptidase and ACE (vasopeptidase inhibition) can prevent functional and morphological features of nephropathy in the Zucker diabetic fatty (ZDF) rat, an animal model of type II diabetes. METHODS: Homozygous (fa/fa) ZDF rats (each n=15) aged 10 weeks were treated with placebo, ramipril (1 mg/kg/day in drinking water), or the vasopeptidase inhibitor AVE7688 (45 mg/kg/day in chow). Metabolic parameters and renal function (metabolic cages) were assessed at baseline (age 10 weeks), and at age 17, 27, and 37 weeks. Twenty heterozygous animals (fa/-) served as lean, nondiabetic controls. At age 37 weeks, the animals were sacrificed and the kidneys analyzed histopathologically. RESULTS: Overt diabetes mellitus (blood glucose >20 mmol/l) was established at age 17 weeks in all homozygous ZDF rats. In the placebo group, urinary protein excretion increased progressively from 8+/-1 (baseline) to 342+/-56 mg/kg/day (week 37) whereas diabetes and proteinuria were absent in the lean control group. Ramipril tended to reduce albuminuria and morphological damage (p=ns) but AVE7688 virtually prevented albuminuria (33+/-12 mg/kg/day, p<0.05 vs. ZDF placebo) and drastically reduced the incidence and severity of glomerulosclerosis and tubulointerstitial damage. CONCLUSIONS: In ZDF rats, development of diabetes mellitus is accompanied by functional and morphological kidney damage that resembles human diabetic nephropathy. Diabetic nephropathy can be prevented by chronic vasopeptidase inhibition.     

Vascular and neural dysfunction in Zucker diabetic fatty rats: a difficult condition to reverse

Aim: We had previously demonstrated that vascular and neural dysfunction in Zucker diabetic fatty (ZDF) rats is progressive. In this study, we sought to determine whether monotherapy of ZDF rats can reverse the vascular and nerve defects. Methods: ZDF rats at 16 weeks of age were treated for 12 weeks with the angiotensin‐converting enzyme inhibitor enalapril, the antioxidant α‐lipoic acid, the HMG‐CoA reductase inhibitor rosuvastatin or the PPARγ agonist rosiglitazone. Vasodilation of epineurial arterioles was measured by videomicroscopy. Endoneurial blood flow (EBF) was measured by hydrogen clearance, and nerve conduction velocity was measured following electrical stimulation of motor or sensory nerves. Results: Motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) (70 and 77% of control, respectively), EBF (64% of control), and vascular relaxation in response to acetylcholine (50% of control) and calcitonin gene‐related peptide (CGRP; 73% of control) are impaired in ZDF rats at 28 weeks of age compared with lean littermate controls. Treatment with enalapril and α‐lipoic acid attenuated the decrease in MNCV and SNCV. Enalapril, α‐lipoic acid and rosiglitazone treatment of ZDF rats were partially effective in improving endothelium‐dependent vascular dysfunction as measured by vascular relaxation in response to acetylcholine. The same drugs also attenuated the decrease in EBF. However, impairment in vascular relaxation in response to CGRP was improved with only α‐lipoic acid or rosuvastatin treatment. The increase in superoxide and nitrotyrosine levels in vascular tissue was attenuated by all treatments. Conclusions: The efficacy of monotherapy treatment of ZDF rats using different classes of drugs for vascular and neural dysfunction once complications have developed did not achieve expected levels. This could be because of the complex aetiology of vascular and neural disease in type 2 diabetes.     

Increasing levels of dietary homocystine with carotid endarterectomy produced proportionate increases in plasma homocysteine and intimal hyperplasia

PURPOSE: The role that homocysteine may play in post-carotid endarterectomy (CEA) restenosis due to intimal hyperplasia is not well understood. This study was designed to investigate the effects of different levels of dietary homocystine on: (1) plasma homocysteine; (2) post-CEA intimal hyperplasia; and (3) levels of the methyl donor S-adenosylmethionine (SAM) and its counterpart S-adenosylhomocysteine (SAH) in the homocysteine pathway. METHODS: Male rats were fed specialized diets for 2 weeks pre- and post-CEA. Groups included control (0 homocystine added, n=9), 1.5 (1.5 g/kg homocystine added, n=10), 3.0 (3.0 g/kg homocystine added, n=9), and 4.5 (4.5 g/kg homocystine added, n=11). The rats underwent a surgical carotid endarterectomy. Endpoints included; plasma homocysteine, intimal hyperplasia, replicative index using with alpha-SM actin and BrdU, hepatic SAM levels, SAH levels, and the hepatic activities of methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS). RESULTS: Increasing dietary homocystine produced a proportionate increase in plasma homocysteine and an increase in intimal hyperplasia. Regression analysis of plasma homocysteine levels and intimal hyperplasia showed a significant correlation (r=0.71,P=0.003). Plasma homocysteine levels above 15 microM were associated with significant increases in intimal hyperplasia above 6.5% (P=0.04). Elevation of plasma homocysteine levels to moderate levels (5-25 microM) resulted in significant post-CEA intimal hyperplasia. Cellular analysis of the area of intimal hyperplasia in all diet groups showed comparable amounts of cells positive for alpha-SM actin. However, with increasing levels of dietary homocystine and plasma homocysteine there was an increase in replicative index (P<0.001) as determined by BrdU staining. Increasing dietary homocystine increased plasma homocysteine and was followed by increases in the replicative index thus producing increased intimal hyperplasia and lumenal stenosis. In hepatic measurements the 1.5 and 3.0 g/kg homocystine diets caused: increased liver activity of MTHFR (P=0.03) and decreased hepatic levels of SAM, SAH and SAM/SAH ratios compared to controls. Homocystine treatment did not cause significant alterations in CBS levels (P=0.992). These studies also showed no correlation of the MTHFR and CBS enzymes with plasma homocysteine levels or intimal hyperplasia. However, hepatic levels of SAM showed significant negative correlations with plasma homocysteine (r=-0.58; P=0.006) and with BrdU percentages of cellular proliferation (r=-0.69; P=0.06). CONCLUSION: The degree of post-CEA intimal hyperplasia in a rat model is directly related to the plasma level of homocysteine. The hyperplastic effects of homocysteine may be mediated in part by a physiological insufficiency of methyl donors as shown by decreases in SAM. Thus, increasing levels of plasma homocysteine enhanced and accelerated the smooth muscle cell response after CEA which led to increased intimal hyperplasia and lumenal stenosis.     

Beneficial effects of melatonin on obesity and lipid profile in young Zucker diabetic fatty rats

Abstract: The study objective was to investigate the effects of melatonin on obesity and obesity‐associated systolic hypertension and dyslipidemia in young male Zucker diabetic fatty (ZDF) rats, an experimental model of the metabolic syndrome. ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups (n = 10): naive (N), vehicle‐treated (V), and melatonin‐treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Melatonin reduced mean weight gain (51 ± 2/100 g BW) versus N‐ZDF group (58 ± 3, P < 0.05) without food intake differences. M‐ZDF rats showed an apparent reduction in systolic hypertension that proved not to be statistically significant, and a significant improvement in dyslipidemia, with a reduction in hypertriglyceridemia from 580 ± 40 to 420.6 ± 40.9 mg/dL (P < 0.01). Melatonin raised high‐density‐lipoprotein (HDL) cholesterol in ZDF (from 81.6 ± 4.9 to 103.1 ± 4.5 mg/dL, P < 0.01) and ZL rats (from 62.8 ± 4.8 to 73.5 ± 4.8 mg/dL, P < 0.05) and significantly reduced low‐density‐lipoprotein (LDL) cholesterol in ZDF rats from 5.20 ± 0.4 to 4.14 ± 0.3 mg/dL (P < 0.05) but had no effect on total cholesterol levels. To our knowledge, this is the first evidence of a positive effect of melatonin on overweight and lipid pattern of obese Zucker diabetic rats, supporting the proposition that melatonin administration may ameliorate overweight and lipid metabolism in humans. Because these benefits occurred in youth, before advanced metabolic and vascular complications, melatonin might help to prevent cardiovascular disease associated with obesity and dyslipidemia.     

Melatonin increases brown adipose tissue mass and function in Zücker diabetic fatty rats: implications for obesity control

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Previously we demonstrated that melatonin browns subcutaneous fat in Zücker diabetic fatty (ZDF) rats. Other works pointed to melatonin as a signal that increases brown adipose tissue (BAT) mass and function in rodents. However, direct proof of thermogenic properties (uncoupled mitochondria) of the newly recruited BAT in response to melatonin is still lacking. Therefore, in this work, we investigated if melatonin recruits thermogenic BAT in ZDF rats. Zücker lean (ZL) and ZDF animals were subdivided into two groups, control (C) and treated with oral melatonin (M) for 6 weeks. Mitochondrial mass, activity of citrate synthase (CS), and respiratory chain complexes I and IV were lower in C‐ZDF than in C‐ZL animals (P < .001). Melatonin treatment increased BAT weight in ZDF rats (P < .001). Also, it rose mitochondrial mass (P < .01) and activities of CS and complexes I and IV (P < .001) in both, ZDF and ZL rats. Uncoupling protein 1 (UCP1) mRNA and protein were 50% lower in BAT from obese rats. Also, guanosine diphosphate (GDP) binding was lower in ZDF than in lean rats (P < .01). Melatonin treatment of obese rats restored the expression of UCP1 and GDP binding to levels of lean rats and sensitized the thermogenic response to cold exposure. These data demonstrated that melatonin recruits thermogenic BAT in ZDF rats. This may contribute to melatonin's control of body weight and its metabolic benefits.     

Comparison of the antilipolytic effects of an A1 adenosine receptor partial agonist in normal and diabetic rats

Introduction and Aims: Elevated plasma free fatty acid (FFA) concentrations play a role in the pathogenesis of type 2 diabetes (2DM). Antilipolytic agents that reduce FFA concentrations may be potentially useful in the treatment of 2DM. Our previous observation that CVT‐3619 lowered plasma FFA and triglyceride concentrations in rats and enhanced insulin sensitivity in rodents with dietary‐induced forms of insulin resistance suggested that it might be of use in the treatment of patients with 2DM. The present study was undertaken to compare the antilipolytic effects of CVT‐3619 in normal (Sprague Dawley, SD) and Zucker diabetic fatty (ZDF) rats. Results: ZDF rats had significantly higher fat pad weight, glucose, insulin and FFA concentrations than those of SD rats. EC₅₀ values for forskolin‐stimulated FFA release from isolated adipocytes from SD and ZDF rats were 750 and 53 nM, respectively (p < 0.05). Maximal forskolin stimulation of FFA release was significantly (p < 0.01) less in ZDF rats (133 ± 60 μM) compared with SD rats (332 ± 38 μM). EC₅₀ values for isoproterenol to increase lipolysis in adipocytes from SD and ZDF rats were 2 and 7 nM respectively. Maximal isoproterenol‐stimulated lipolysis was significantly (p < 0.01) lower in adipocytes from ZDF rats (179 ± 23 μM) compared with SD rats (343 ± 27 μM). Insulin inhibited lipolysis in adipocytes from SD rats with an IC₅₀ value of 30 pM, whereas adipocytes from ZDF rats were resistant to the antilipolytic actions of insulin. In contrast, IC₅₀ values for CVT‐3619 to inhibit the release of FFA from SD and ZDF adipocytes were essentially the same (63 and 123 nM respectively). CVT‐3619 inhibited lipolysis more than insulin in both SD (86 vs. 46%, p < 0.001) and ZDF (80 vs. 13%, p < 0.001) adipocytes. In in vivo experiments, CVT‐3619 (5 mg/kg, PO) lowered FFA to a similar extent in both groups. Plasma concentrations of CVT‐3619 were not different in SD and ZDF rats. There was no significant difference in the messenger RNA expression of the A₁ receptors relative to β‐actin expression in adipocytes from SD (0.98 ± 0.2) and ZDF rats (0.99 ± 0.3). Conclusion: The antilipolytic effects of CVT‐3619 appear to be independent of insulin resistance and animal model.     

Renal accumulation and clearance of advanced glycation end-products in type 2 diabetic nephropathy: effect of angiotensin-converting enzyme and vasopeptidase inhibition

Aims/hypothesis Renal accumulation of AGEs may contribute to the progression of diabetic nephropathy. We evaluated the effect of ramipril (a pure ACE inhibitor) and AVE7688 (a dual inhibitor of ACE and neutral endopeptidase) on renal accumulation of the advanced glycation end-product (AGE) 3-deoxyglucosone-imidazolone, carboxymethyllysine (CML) and pentosidine, and on clearance of CML in type 2 diabetes.Methods Male Zucker diabetic fatty rats (ZDF, Gmi-fa/fa) rats were treated from age 10 to 37 weeks with ramipril (1 mg·kg^–1·day^–1), AVE7688 (45 mg·kg^–1·day^–1) or without drug. Ramipril and AVE7688 reduced albuminuria by 30 and 90%, respectively.Results ZDF rats showed increased renal accumulation of the AGE subtypes 3-deoxyglucosone-imidazolone, pentosidine and CML by about 40, 55 and 55%, respectively compared with heterozygous, non-diabetic control animals at the age of 37 weeks. AVE7688 but not ramipril attenuated the renal accumulation of 3-deoxyglucosone-imidazolone, pentosidine and CML and improved CML clearance in ZDF rats. During glycation reactions in vitro, AVE7688 also demonstrated potent chelating activity and inhibited metal-catalysed formation of pentosidine and CML.Conclusions/interpretation Improved AGE clearance and direct inhibition of AGE formation by chelation may contribute to reduced accumulation of renal AGEs and to the nephroprotective effects of vasopeptidase inhibition in type 2 diabetes.     

Ischaemic preconditioning does not protect the heart in obese and lean animal models of Type 2 diabetes

Aims/hypothesis The prevalence of Type 2 diabetes mellitus is increasing worldwide with obese diabetic patients constituting the majority of this population. Type 2 diabetes is associated with increased morbidity and mortality after acute myocardial infarction. Previous experimental studies of ischaemia-reperfusion tolerance in diabetes have only been performed in animal models of Type 1 diabetes mellitus, yielding conflicting data. The aim of the present study was to characterise and compare the tolerance to ischaemia and effects of ischaemic preconditioning (IPC) in hearts from obese Zucker diabetic fatty (ZDF) and lean Goto-Kakizaki (GK) Type 2 diabetic rats, using non-obese Zucker and Wistar rats as respective controls.Methods The two rat strains were divided into 8 groups. The ZDF study (n=47) consisted of: Control –IPC, Control +IPC, ZDF –IPC and ZDF +IPC. The GK study (n=38) consisted of: Control –IPC, Control +IPC, GK –IPC and GK +IPC. Hearts, which were studied in a Langendorff preparation perfused with Krebs-Henseleit buffer, were subjected or not to IPC (+IPC, –IPC) before 50 minutes of regional ischaemia and 120 minutes reperfusion.Results Ischaemic reperfusion injury was smaller in obese (p<0.05) and lean (p<0.05) Type 2 diabetic animals than in their respective control animals. IPC reduced ischaemic reperfusion injury during reperfusion in non-diabetic control rats (p<0.01), but failed to protect hearts from both diabetic animal models. Post-ischaemic haemodynamic recovery was impaired in the ZDF rats compared to both control and GK rats (p<0.05).Conclusions/interpretation Ischaemic preconditioning does not protect hearts from obese or lean Type 2 diabetic animals. However, the susceptibility of the Type 2 diabetic myocardium to ischaemic damage is lower than in non-diabetic hearts. The method described here could be used as a tool to study the pathogenesis of increased cardiovascular morbidity and mortality in Type 2 diabetes.     

The equivocal metabolic response to endotoxaemia in type 2 diabetic and obese ZDF rats

Aims/hypothesis The metabolic and endocrine disturbances associated with obesity and type 2 diabetes may impair the normal metabolic response to injury. Our objective was to investigate amino acid metabolism in endotoxaemic type 2 diabetic obese rats.Materials and methods A metabolic study was performed over 4 days using male Zucker diabetic fatty (ZDF) rats (fa/fa) and lean littermates (fa/+) divided into three groups: ad libitum-fed groups which underwent no treatment, lipopolysaccharide (LPS)-treated groups receiving E. coli LPS by i.p. injection, and pair-fed groups to the respective LPS groups. We evaluated the effect of endotoxaemia on body weight, food intake and tissue weights. Nitrogen loss and muscular proteolysis were measured daily by determination of urinary 3-methylhistidine (3-MH) excretion. Plasma, intestine and muscle amino acid levels were measured.Results The data showed that ad libitum-fed ZDF rats had lower plasma arginine and glutamine levels than ad libitum-fed control rats. Compared with control rats, the LPS-treated ZDF rats presented lower thymic involution, a lower 3-MH:creatinine ratio and higher cumulative nitrogen balance.Conclusions/interpretation Against our working hypothesis, ZDF rats did not show an impaired metabolic response, and even appeared to be less sensitive to the stress.L. Belabed and G. Senon made an equal contribution to this study.