Effects of the dual PPAR-α/γ agonist aleglitazar on glycaemic control and organ protection in the Zucker diabetic fatty rat.

AIMS: To evaluate the effects of aleglitazar, a dual peroxisome proliferator-activated receptor-α/γ agonist, on the development of diabetes-related organ dysfunction, in relation to glycaemic and lipid changes, in Zucker diabetic fatty (ZDF) rats. METHODS: Six-week-old, male ZDF rats received aleglitazar 0.3 mg/kg/day or vehicle as food admix for 13 weeks (n = 10 per group). Age-matched male Zucker lean rats served as non-diabetic controls. Plasma and renal markers were measured at several time points. Histopathology and quantitative immunohistochemistry were performed at 13 weeks. RESULTS: Glycated haemoglobin (5.4 vs. 9.2%) and blood glucose (8.3 ± 0.3 vs. 26.1 ± 1.0 mmol/l) were significantly reduced at 12 weeks with aleglitazar versus vehicle-treated ZDF rats (both p < 0.01), while aleglitazar preserved near-normal plasma insulin levels. Aleglitazar prevented the development of hypertriglyceridaemia (1.4 ± 0.1 vs. 8.5 ± 0.9 mmol/l) and reduced plasma non-esterified fatty acids (0.09 ± 0.02 vs. 0.26 ± 0.04 mmol/l) relative to vehicle-treated animals (both p < 0.01). Urinary glucose and protein concentrations were significantly reduced at 13 weeks with aleglitazar versus vehicle-treated rats (both p < 0.01). Consistent with its effect on glycaemic control, aleglitazar protected ß-cell morphology, as evidenced by preservation of islet integrity, and reduction of ß-cell apoptosis and islet fibrosis. Aleglitazar prevented renal glomerular hypertrophy, podocyte degeneration, glomerulosclerosis, tubulo-interstitial lesions and development of cataracts. CONCLUSIONS: Aleglitazar strongly improved glycaemic and lipid parameters while protecting key tissues, including the pancreas, kidneys and eyes, against diabetes-associated structural and functional changes in the ZDF rat.

Taspoglutide, a novel human once-weekly GLP-1 analogue, protects pancreatic ß-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 ß-cells. We evaluated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, on ß-cells in vitro and in vivo. METHODS: Proliferation of murine pancreatic ß (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-1ß, 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 ß-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 ß-cell proliferation, prevented apoptosis in vitro and exerted multiple ß-cell protective effects on islet architecture and function in vivo in ZDF rats.

Taspoglutide, a novel human once-weekly analogue of glucagon-like peptide-1, improves glucose homeostasis and body weight in the Zucker diabetic fatty rat.

AIM: Glucagon-like peptide-1 (GLP-1) receptor agonists are a novel class of pharmacotherapy for type 2 diabetes. We investigated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, in the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. METHODS: Blood glucose and plasma levels of insulin, peptide YY (PYY), glucose-dependent insulinotropic polypeptide (GIP) and triglycerides were measured during oral glucose tolerance tests (oGTT) conducted in ZDF rats treated acutely or chronically with a single long-acting dose of taspoglutide. Pioglitazone was used as a positive control in the chronic study. Postprandial glucose, body weight, glycaemic control and insulin sensitivity were assessed over 21 days in chronically treated animals. RESULTS: Acute treatment with taspoglutide reduced glucose excursion and increased insulin response during oGTT. In chronically treated rats, glucose excursion and levels of GIP, PYY and triglycerides during oGTT on day 21 were significantly reduced. Postprandial glucose levels were significantly lower than vehicle controls by day 15. A significant reduction in body weight gain was noticed by day 8, and continued until the end of the study when body weight was approximately 7% lower in rats treated with taspoglutide compared to vehicle. Glycaemic control (increased levels of 1,5-anhydroglucitol) and insulin sensitivity (Matsuda index) were improved by taspoglutide treatment. CONCLUSIONS: Taspoglutide showed typical effects of native GLP-1, with improvement in glucose tolerance, postprandial glucose, body weight, glycaemic control and insulin sensitivity.

High prevalence of prediabetes and diabetes in a population exposed to high levels of an organochlorine cocktail.

AIMS/HYPOTHESIS: A heavily polluted area of Eastern Slovakia was targeted by the PCBRISK cross-sectional survey to search for possible links between environmental pollution and both prediabetes and diabetes. METHODS: Associations of serum levels of five persistent organic pollutants (POPs), namely polychlorinated biphenyls (PCBs), 2,2'-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), 2,2'-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p'-DDT), hexachlorobenzene (HCB) and beta-hexachlorocyclohexane (beta-HCH), with prediabetes and diabetes were investigated in 2,047 adults. Diabetes and prediabetes were diagnosed by fasting plasma glucose in all participants and by OGTT in 1,220 compliant participants. RESULTS: Our population was stratified in terms of individual POPs quintiles and associations between environmental pollution, prediabetes and diabetes were investigated. Prevalence of prediabetes and diabetes increased in a dose-dependent manner, with individuals in upper quintiles of individual POPs showing striking increases in prevalence of prediabetes as shown by OR and 95% CI for PCBs (2.74; 1.92-3.90), DDE (1.86; 1.17-2.95), DDT (2.48; 1.77-3.48), HCB (1.86; 1.7-2.95) and beta-HCH (1.97; 1.28-3.04). Interestingly, unlike PCBs, DDT and DDE, increased levels of HCB and beta-HCH seemed not to be associated with increased prevalence of diabetes. Nevertheless, individuals in the 5th quintile of the variable expressing the cumulative effect of all five POPs (sum of orders) had a more than tripled prevalence of prediabetes and more than six times higher prevalence of diabetes when compared with the 1st referent quintile. CONCLUSIONS/INTERPRETATION: Increasing serum concentrations of individual POPs considerably increased prevalence of prediabetes and diabetes in a dose-dependent manner. Interaction of industrial and agricultural pollutants in increasing prevalence of prediabetes or diabetes is likely.

Reduced plaque formation induced by rosiglitazone in an STZ-diabetes mouse model of atherosclerosis is associated with downregulation of adhesion molecules.

Adhesion molecules have been implicated in the development and progression of cardiovascular disease, which is highly prevalent in people with diabetes. Adhesion molecules can mediate adhesion of leukocytes to the endothelium. Furthermore, P-selectin expressed on platelets is able to mediate the adhesion of leukocytes to platelets. In this study, we examine the in-vivo and in-vitro effects of rosiglitazone with particular emphasis on three important adhesion molecules (VCAM-1, ICAM-1 and P-selectin). In the aorta of STZ-diabetic apolipoprotein E-deficient (apoE KO) mice, rosiglitazone significantly reduced both total and arch plaque area. The mechanism for this appeared to be reduced macrophage infiltration into the atherosclerotic plaque which was also associated with reduced mRNA levels for VCAM-1, ICAM-1, MCP-1 and P-selectin in the aorta. In-vitro studies revealed reduced cell adhesion of monocytic cells (THP-1) to fibrinogen and endothelial cells (HUVEC) after incubation with rosiglitazone. Furthermore, the reduction in leukocyte adhesion also correlated with significant reductions in mRNA levels for VCAM-1, ICAM-1 and P-selectin indicating that reduced macrophage infiltration in atherosclerotic plaques may occur as a result of a direct effect of rosiglitazone on adhesion molecules in both monocytes and endothelial cells. Thus, we have shown that rosiglitazone appears to have direct anti-atherosclerotic effects in an animal model of diabetes-associated atherosclerosis which are at least partly due to effects on VCAM-1, ICAM-1, MCP-1 and P-selectin expression which leads to decreased leukocyte adhesion and macrophage infiltration.

Concerted action of leptin in regulation of fatty acid oxidation in skeletal muscle and liver.

Central action of leptin on food intake and energy expenditure is integrated with leptin's peripheral action modulating the fatty acid and glucose metabolism and preventing the accumulation of lipids in nonadipose tissues. However, exact mechanism(s) of the leptin's action in the peripheral tissues has not yet been fully elucidated. Therefore, we investigated the effect of a single intravenous injection of leptin on palmitoyl-CoA and palmitoyl-carnitine oxidation rate in liver and skeletal muscle followed by measurements of the carnitine-palmitoyl transferase 1 (CPT1) activity and activities of ss-oxidation enzymes in mitochondria (acyl-CoA dehydrogenase) and in peroxisomes (acyl-CoA oxidase) of rats. Animals were euthanized and tissues and serum harvested 15 min, 1 hour, 3 hours and 6 hours after leptin administration. Intravenous leptin injection increased mitochondrial palmitoyl-CoA oxidation rate in both liver (95%; P<0.025) and skeletal muscle (2.7-fold; P<0.05). This was paralleled by lowering hepatic (-156%; P<0.001) and skeletal muscle (-191%; P<0.001) triglyceride content. Leptin-induced elevation of palmitoyl-CoA oxidation rate in liver was paralleled by increased CPT1 activity (52%; P<0.05) and ss-oxidation capacity (52%; P<0.05). Lack of the leptin's effect on the CPT1-activity in muscle (20%; p=0.09) suggests the existence of an alternative pathway for increasing the palmitoyl-CoA-oxidation rate bypassing the CPT1 regulatory step. Interestingly, leptin stimulated the overall ss-oxidation capacity in muscle by 69% (P=0.027). This may indicate to an involvement of mitochondrial acyl-CoA dehydrogenases as well as of peroxisomal fat catabolism. Taken together, we showed that leptin acutely increases palmitoyl-CoA oxidation rate in liver and in skeletal muscle, which was associated with tissue specific effect on the CPT1 activity as well as on the downstream enzymes of fatty acid oxidation pathways in rat mitochondria and peroxisomes. Tangible evidence for the leptin-induced increase of fatty acid catabolism was provided by a lowered skeletal muscle and hepatic lipid deposition.

Multiple organochlorine pollution and the thyroid.

OBJECTIVE: Although the biological effects of major persistent organochlorinated pollutants (POPs) appear to be essentially similar, some effects which would be specific for certain substance cannot be excluded. We attempted to study the thyroid volume and thyrotropin level in the population living in the area with multiple pollution by polychlorinated biphenyls (PCBs) and pesticides (DDE and hexachlorobenzene - HCB). METHODS: A total of 454 adults was examined within the pilot field survey in 1998. Among them were 237 males (age range 19-78 years, median 47) and 227 females (age range 19-78 years, median 48). Fifteen environmentally prevalent congeners of polychlorinated biphenyls and also p,p-DDE (2,2-bis(4-chlorophenyl)-1,1-dichloroethylene), p,p-DDT (2,2-bis(4-chlorophenyl)- 1,1,1-trichloro-ethane), hexachlorobenzene (HCB) as well as alpha-, beta- and gamma-hexachlorocyclohexane (HCH) were determined in serum by high resolution gas chromatography using microelectron capture detector and microcapillary column. Thyroid volume (ThV) was measured by real time sonography using the ellipsoid method with the aid of sonographic instrument Sonoline SI-400 (Siemens, Germany). The level of TSH was estimated by supersensitive immunoradiometric method using commercial kits by Immunotech (Marseille, France). Pearsons correlation coefficients after logarithmic transformation of values and Spearmans correlation coefficients were used for statistical evaluation. RESULTS: Significant positive association (p<0.01) was found between DDE and PCB, DDE and HCB, while that between PCB and HCB was not significant. Similar positive association (p<0.01) was also found between each individual organochlorine and their sum. Significant negative association (p<0.01) was found between ThV and TSH. When using categorical PCB values either >2000 (N=208) or >3000 (N=127) ng/g lipid, significant positive association (p<0.05 and p<0.01, respectively) was found between the sum of all organochlorines (PCB+DDE+HCB) and ThV, while that between PCB and ThV (p<0.01) was found only at the PCB levels >3000 ng/g lipid. When using Spearmans correlation coefficients, significant negative association appeared between PCB and TSH (p<0.05), sum of organochlorines and TSH (p<0.05) and ThV and TSH (p<0.01). CONCLUSIONS: Although several significant positive and negative associations were found, this study, like several others, could not exactly define the participation level of individual POPs in their common toxic effects, but possibly contributed to the recognition and elucidation of some problems related to this task.

Insulin sensitivity indices: a proposal of cut-off points for simple identification of insulin-resistant subjects.

Demanding measurement of insulin sensitivity using clamp methods does not simplify the identification of insulin resistant subjects in the general population. Other approaches such as fasting- or oral glucose tolerance test-derived insulin sensitivity indices were proposed and validated with the euglycemic clamp. Nevertheless, a lack of reference values for these indices prevents their wider use in epidemiological studies and clinical practice. The aim of our study was therefore to define the cut-off points of insulin resistance indices as well as the ranges of the most frequently obtained values for selected indices. A standard 75 g oral glucose tolerance test was carried out in 1156 subjects from a Caucasian rural population with no previous evidence of diabetes or other dysglycemias. Insulin resistance/sensitivity indices (HOMA-IR, HOMA-IR2, ISI Cederholm, and ISI Matsuda) were calculated. The 75th percentile value as the cut-off point to define IR corresponded with a HOMA-IR of 2.29, a HOMA-IR2 of 1.21, a 25th percentile for ISI Cederholm, and ISI Matsuda of 57 and 5.0, respectively. For the first time, the cut-off points for selected indices and their most frequently obtained values were established for groups of subjects as defined by glucose homeostasis and BMI. Thus, insulin-resistant subjects can be identified using this simple approach.

Human thyroid in the population exposed to high environmental pollution by organochlorinated pollutants for several decades.

OBJECTIVE: To study possible effects of long-time exposure of chemical factory employees and population of surrounding polluted area to polychlorinated biphenyls and pesticides on the thyroid volume and function as compared to the population from the area of background pollution. METHODS: A total of 461 adults consisting of 239 men and 222 women was examined and divided into four groups according to their permanent domicile as related to the level of environmental pollution, e.g. SR (area of background pollution, n = 207), SI (slightly polluted area, n = 59), MI (polluted city of Michalovce, n = 94) and CH (employees of chemical factory subjected to high PCB exposure, n = 101), combined first three groups being also called LPA (less polluted areas, n = 360). Thyroid volume (ThV) and echogenicity were measured by real time sonography. The level of polychlorinated biphenyls (PCB) and pesticides (hexachlorbenzene--HCB, DDE (2,2'-2-bis(4-chlorobiphenyl)- 1,1-dichloroethylene), p,p'-DDT (2,2'-bis(4-chlorophenyl)- 1,1,1-trichloroethane) and alpha-, beta- and gamma-hexachlorcyclohexane--HCH) was estimated by congener specific analysis using HP 5890 gas chromatograph with a 63Ni electron capture detector. Serum levels of thyrotropin (TSH) and thyroid peroxidase antibodies (anti-TPO) were measured by specific sensitive immunoassays. RESULTS: The association of very high PCB level (e.g. 7300 +/- 871 ng/g lipid; mean +/- S.E.) with increased ThV (e.g. 16.3 +/- 0.73 ml) in CH has been found, the values being significantly higher than these of 360 subjects in LPA (e.g. 2045 +/- 147 ng/g, p < 0.001 for PCB and 14.0 +/- 0.32 ml, p < 0.001 for ThV). In 23 subjects from CH with PCB level > 10000 ng/g the ThV was 18.7 +/- 2.32 ml, while that in 251 subjects from LPA with PCB level of < 2000 ng/g was 13.8 +/- 0.35 ml (p < 0.05). In addition, ThV as well as PCB levels were strikingly increasing with age. In parallel with PCB levels, also the levels of other organochlorines estimated (namely these of DDE) were increasing. Although the participation of these substances in the development of adverse effects cannot yet be defined, it cannot be excluded. The association of increased levels of episodic congener PCB 101 with increased ThV appeared to be more pronounced than that of stable congeners PCB 153 and 180. Finally, significant increase in the frequency of thyroid hypoechogenicity by ultrasound, ThV > 20.0 ml and thyroperoxidase antibodies in CH area was observed as compared to LPA. CONCLUSIONS: Several associations of high PCB and pesticides level with characteristics of thyroid disorders (e.g. increased thyroid volume, frequency of hypoechogenicity and frequency of positive thyroperoxidase antibodies level in blood) were observed in the area with heavy industrial pollution by PCB.

Mapping of genetic determinants of the sympathoneural response to stress.

Activation of the sympathoadrenal system (SAS, comprising the sympathetic nervous system and the adrenal medulla) in response to stressful stimuli is an important defense mechanism as well as a contributor to several cardiovascular diseases. There is variability in the SAS response to stress, although the extent to which this is genetically regulated is unclear. Some rodent models, including the hereditary hypertriglyceridemic (hHTg) rat, are hyperresponsive to stress. We investigated whether quantitative trait loci (QTLs) that affect sympathoadrenal response to stress could be identified. Second filial generation rats (n = 189) derived from a cross of the hHTg rat and the Brown Norway rat had plasma norepinephrine (NE) and epinephrine (Epi) levels, indices of activation of the sympathoneural and adrenal medulla components, respectively, measured in the resting state and in response to an immobilization stress. Responses were assessed early (20 min) and late (120 min) after the application of the stress. A genome scan was conducted using 153 microsatellite markers. Two QTLs (maximum peak LOD scores of 4.17 and 3.52, respectively) influencing both the early and late plasma NE response to stress were found on chromosome 10. Together, the QTLs accounted for approximately 20% of the total variation in both the early and late NE responses in the F(2) rats. Interestingly, the QTLs had no effect on plasma Epi response to stress. These findings provide evidence for a genetic determination of the response of a specific component of the SAS response to stress. Genetically determined variation in sympathetic nervous system response to stress may contribute to cardiovascular diseases.

The hypotriglyceridemic effect of dietary n-3 FA is associated with increased beta-oxidation and reduced leptin expression.

To study the mechanisms responsible for the hypotriglyceridemic effect of marine oils, we monitored the effects of high dietary intake of n-3 PUFA on hepatic and muscular beta-oxidation, plasma leptin concentration, leptin receptor gene expression, and in vivo insulin action. Two groups of male Wistar rats were fed either a high-fat diet [28% (w/w) of saturated fat] or a high-fat diet containing 10% n-3 PUFA and 18% saturated fat for 3 wk. The hypotriglyceridemic effect of n-3 PUFA was accompanied by increased hepatic oxidation of palmitoyl-CoA (125%, P < 0.005) and palmitoyl-L-carnitine (480%, P < 0.005). These findings were corroborated by raised carnitine palmitoyltransferase-2 activity (154%, P < 0.001) and mRNA levels (91%, P < 0.01) as well as by simultaneous elevation of hepatic peroxisomal acyl-CoA oxidase activity (144%, P < 0.01) and mRNA content (82%, P < 0.05). In contrast, hepatic carnitine palmitoyltransferase-1 activity remained unchanged despite a twofold increased mRNA level after n-3 PUFA feeding. Skeletal muscle FA oxidation was less affected by dietary n-3 PUFA, and the stimulatory effect was found only in peroxisomes. Dietary intake of n-3 PUFA was followed by increased acyl-CoA oxidase activity (48%, P < 0.05) and mRNA level (83%, P < 0.05) in skeletal muscle. The increased FA oxidation after n-3 PUFA supplementation of the high-fat diet was accompanied by lower plasma leptin concentration (-38%, P < 0.05) and leptin mRNA expression (-66%, P < 0.05) in retroperitoneal adipose tissue, and elevated hepatic mRNA level for the leptin receptor Ob-Ra (140%, P < 0.05). Supplementation of the high-fat diet with n-3 PUFA enhanced in vivo insulin sensitivity, as shown by normalization of the glucose infusion rate during euglycemic hyperinsulinemic clamp. Our results indicate that the hypotriglyceridemic effect of dietary n-3 PUFA is associated with stimulation of FA oxidation in the liver and to a smaller extent in skeletal muscle. This may ameliorate dyslipidemia, tissue lipid accumulation, and insulin action, in spite of decreased plasma leptin level and leptin mRNA in adipose tissue.

From naturally occurring goitrogens to the effects of anthropogenic endocrine disruptors on the thyroid in Slovakia.

The objective is to describe the effect of mandatory and well monitored prophylaxis of endemic goitre in Slovakia with the use of iodised salt for past 50 years and previous experimental attempts to assess a possible participation of naturally occurring goitrogens in the etiology of goitre. Previous observations by others showed a striking decrease of goitre prevalence in schoolchildren as early as few years after starting the prophylaxis in Slovakia and this success has been confirmed by a European study conducted by others in mid nineties. In the meantime, however, massive industrialisation of Slovakia and environmental negligence of the administration resulted in heavy airborne and mainly waterborne pollution of the food chain in certain areas around large chemical factories by industrial waste including polychlorinated biphenyls (PCB). Since Slovakia has been recently evaluated as an iodine replete country as based on the findings of the low thyroid volume by ultrasound and high urinary iodine, it became possible to evaluate the participation of factors different from iodine deficiency in the thyroid growth. Recent field surveys including an extensive international study PCBRISK repeatedly showed highly increased blood PCB level in subjects living in the polluted area and, at the same time, increased thyroid volume by ultrasound, increased frequency of positive thyroid antibodies and impaired glucose tolerance. (Ref. 59)

Iodine deficiency in vegetarians and vegans.

Iodine content in food of plant origin is lower in comparison with that of animal origin due to a low iodine concentration in soil. Urinary iodine excretion was assessed in 15 vegans, 31 lacto- and lacto-ovovegetarians and 35 adults on a mixed diet. Iodine excretion was significantly lower in alternative nutrition groups - 172 microg/l in vegetarians and 78 microg/l in vegans compared to 216 microg/l in subjects on a mixed diet. One fourth of the vegetarians and 80% of the vegans suffer from iodine deficiency (iodine excretion value below 100 microg/l) compared to 9% in the persons on a mixed nutrition. The results show that under conditions of alternative nutrition, there is a higher prevalence of iodine deficiency, which might be a consequence of exclusive or prevailing consumption of food of plant origin, no intake of fish and other sea products, as well as reduced iodine intake in the form of sea salt.

Mapping of genetic loci predisposing to hypertriglyceridaemia in the hereditary hypertriglyceridaemic rat: analysis of genetic association with related traits of the insulin resistance syndrome.

AIMS/HYPOTHESIS: Hypertriglyceridaemia is an important risk factor for coronary heart disease, especially in the context of the insulin resistance syndrome where it often occurs with hypertension. The two phenotypes are also associated in the hereditary hypertriglyceridaemic (hHTg) rat. The aim of this study was to map quantitative trait loci that affect plasma triglyceride concentration in the hHTg rat and determine whether they co-localize with loci for blood pressure. METHODS: Second filial generation progeny (n=189) from a cross of the hHTg rat with the Brown Norway rat were phenotyped for fasting plasma triglyceride, glucose and insulin concentrations, and direct unrestrained resting blood pressure. A partial genome-scan was conducted using 153 microsatellite markers that were polymorphic between the two strains. RESULTS: A major locus (lod score 6.5) influencing plasma triglyceride concentration in a co-dominant fashion was mapped to chromosome 4 between D1Mit 5 and D1Mit17. Chromosome 8 contained multiple peaks with a lod score greater than 4.0 influencing triglyceride concentration. Importantly, none of the triglyceride loci had an effect on blood pressure. The triglyceride locus on chromosome 4 co-localized with a locus for fasting plasma insulin (lod score 4.1), although the effect on insulin concentration was in the opposite direction to that on triglyceride. CONCLUSION/INTERPRETATION: We have mapped the major loci that affect plasma triglyceride concentration in the hHTg rat. These loci do not influence blood pressure suggesting that these commonly associated phenotypes of the insulin resistance syndrome are not be due to pleiotropic effects of the same gene(s).

Thyroid function and cholesterol level: paradoxical findings in large groups of population with high cholesterol food intake.

OBJECTIVE: To compare the levels of serum cholesterol with thyroid function as estimated by the level of thyrotropin and free thyroxine with possible participation of thyroperoxidase antibodies in large number of adults examined within large field surveys focused on the evaluation of thyroid status of Slovak rural population. SUBJECTS AND METHODS: Serum level of cholesterol and thyrotropin (TSH) was estimated in a total of 2786 adults. In addition, in 2038 of them also the level of free thyroxine (FT4), total triiodothyronine (TT3), cholesterol, triglycerides and phospholipids was measured. The levels of TSH, anti-TPO and FT4 were estimated by supersensitive electrochemiluminiscent immunoassay using the automatic system Elecsys (Roche, Switzerland). RESULTS: A total of 2786 adults was stratified into 7 groups according to the range of TSH level as related to generally recognized level of thyroid function, e.g. 1. TSH <0.10 mU/L (overt hyperthyroidism, N=41), 2. TSH 0.11-0.30 mU/L (overt or subclinical hyperthyroidism, N=149), 3. TSH 0.31-2.50 mU/L (normal level, N=1750), 4. TSH 2.51-4.50 ("high normal" level, N=607), 5 TSH 4.51-6.50 (mild or incipient subclinical hypothyroidism, N=137), 6. TSH 6.51-10.00 mU/L (mild hypothyroidism, N=50), 7. TSH 10.01-99.00 mU/L (severe hypothyroidism, N=53). The average levels of cholesterol in all groups were very similar ranging from 5.53 to 6.17 mmol/L and no interrelations with TSH level were found. In addition, no considerable differences between these groups were found when considering the levels of medians, upper quartiles and 90th percentiles of individual groups. When male and female subjects were divided into age groups according to the decades, an age dependent increase of cholesterol level was found in both sexes. The fraction of 2038 subjects was divided into the same TSH related groups as defined above. Similarly as above, no considerable differences in cholesterol, triglycerides and phospholipids level were observed. However, the levels of FT4 and TT3 were significantly decreasing with the increase of TSH level which confirmed the continuing decrease of thyroid function. The frequency of positive anti-TPO in subjects with TSH >6.5 mU/l (71/86 = 82.5%) was significantly higher than that in subjects with TSH <6.5 mU/l (468/1952 = 23.9%). CONCLUSIONS: No difference in the level of cholesterol and triglycerides was found in large groups of rural adults from Slovakia with various thyroid function as estimated by the level of TSH, FT4, TT3 and anti-TPO. It is assumed that this interrelation resulted from very high cholesterol intake due to inappropriate general nutritional status of rural population resulting from the consumption of unhealthy foods.

Comparison of the extrapancreatic action of gamma-linolenic acid and n-3 PUFAs in the high fat diet-induced insulin resistance [corrected].

OBJECTIVE: The effect of dietary borage oil (rich in the gamma-linolenic acid [GLA]) on insulin sensitivity and lipid metabolism was compared with that of fish oil (rich in n-3 polyunsaturated fatty acids [PUFAs]) in high fat (HF) diet-induced insulin resistance (IR) of rats. METHODS: Male Wistar rats were fed ad libitum for 3 weeks a standard laboratory chow (Controls) or high fat diet consisting of 70-cal % fat. In addition, a group of rats was fed high fat (HF) diet where a part of saturated fat was replaced with fish oil as a source of n-3 PUFAs (HF+FO), or borage oil as a source of GLA (HF+GLA). In vivo insulin action was assessed by the euglycemic hyperinsulinemic clamp. Glucose, insulin, free fatty acids (FFA), triglycerides (Tg) and glycerol levels in blood and tissue depots were also measured. RESULTS: Increased levels of Tg, FFA and glycerol in circulation after HF diet were accompanied by their raised accumulation in insulin sensitive tissues. FO feeding lowered the concentration of all lipids in serum and prevented their accumulation in both tissues. On the other hand GLA supplementation into the high fat diet did not suppress increased levels of Tg, FFA and glycerol in circulation and tissue depots as well. FO feeding significantly reduced HF diet-induced in vivo IR, while GLA supplementation did not improve the in vivo insulin sensitivity in HF diet induced insulin resistance. CONCLUSIONS: 1. Substitution of FO into the high fat diet led to an improvement of in vivo insulin action; 2. this insulin sensitizing effect of FO was accompanied by a decrease of circulating Tg, FFA and glycerol levels in the postprandial state and by a lower lipid content in liver and skeletal muscle. 3. on the opposite, GLA treatment failed to improve in vivo insulin action; and 4. was associated with an adverse effect on lipid levels both in circulation and tissue depots.

An increase in peroxisomal fatty acid oxidation is not sufficient to prevent tissue lipid accumulation in hHTg rats.

We observed earlier that increased skeletal muscle lipid content in the hereditary hypertriglyceridemic (hHTg) rat is accompanied by a decline in plasma leptin. Leptin has recently been shown to enhance peripheral insulin sensitivity by decreasing the tissue triglyceride accumulation, possibly through regulation of fatty acid oxidation and lipogenesis. Thus, to test the hypothesis that insulin resistance and increased skeletal muscle lipid accumulation in hHTg rats are due to a defect in lipid catabolism, we measured mitochondrial and peroxisomal fatty acid oxidation and malonyl-CoA and acetyl-CoA carboxylase-2 content in skeletal muscles of these animals. In addition, we investigated possible molecular mechanisms responsible for the lower leptin levels in hHTg rats by measuring leptin and leptin-receptor (Ob-Ra) mRNA levels. We found the following: (1) in spite of a higher skeletal muscle malonyl-CoA content and an increased sensitivity of carnitine palmitoyltransferase-1 to malonyl-CoA, carnitine palmitoyltransferase-1 activity in muscle of hHTg rats was normal; (2) increased peroxisomal fatty acid oxidation did not seem to be sufficient to prevent the tissue lipid accumulation in these animals; (3) both lower leptin production by white adipose tissue and increased leptin uptake seem to be responsible for lower circulating leptin levels and therefore lower fatty acid catabolism.

Insulin resistance in the hereditary hypertriglyceridemic rat is associated with an impairment of delta-6 desaturase expression in liver.

Our previous studies have shown that insulin resistance (IR) in the hereditary hypertriglyceridemic (hHTg) rat is accompanied by a specific fatty acid (FA) profile in insulin target tissues, possibly due to a defect in the desaturation pathway. Increased dietary intake of n-3 polyunsaturated fatty acids (PUFAs) was shown to shape FA composition and to improve insulin sensitivity in this animal strain. Thus, the aim of this study is twofold: (1) to evaluate a defect in the FA desaturation by direct measurement of enzyme activity and gene expression for Delta-6 desaturase (Delta-6 D) in liver of hHTg rats and (2) to investigate the effect of dietary n-3 PUFAs on hepatic Delta-6 D in relation to tissue FA composition. Male Wistar or hHTg rats were fed ad libitum for 21 days either the basal or fish oil (FO)-supplemented diets. Triglyceride (Tg) levels in serum and tissue lipid extracts were measured with the aid of a commercially available enzymatic set. Hepatic activity of the Delta-6 D was determined radiometrically in a microsomal fraction using 1-(14)C-linoleic acid as a substrate. The Delta-6 D mRNA levels were measured using the Northern blot technique. Tissue FA composition was determined by gas chromatography in the total phospholipid fraction after TLC separation. Increased levels of Tg in hHTg rat circulation were accompanied by raised accumulation of Tg in skeletal muscles. FO feeding lowered the concentration of Tg in serum and prevented their accumulation in skeletal muscles of hHTg rats. A pronounced decrease in the hepatic Delta-6 D activity in hHTg rats (by about 80%) was not further diminished by FO feeding. On the other hand, the activity of Delta-6 D in liver of control rats was reduced by about 40% after FO supplementation. These changes were paralleled by a decrease in the Delta-6 D index as calculated from the liver phospholipid FA profile. In particular, an increase in the amount of 18:2 n-6 and a decrease in arachidonic acid and PUFA n-6 metabolites were found. The results indicate that a decrease of insulin action in hHTg rats is accompanied by an impairment of the hepatic Delta-6 D activity already at the gene level, which is not further affected by n-3 PUFA supplementation.

Lipids and insulin resistance: what we've learned at the Fourth International Smolenice Symposium.

A summary of the Fourth International Smolenice Symposium on Lipids and Insulin Resistance focusing on "The Role of Fatty Acid Metabolism and Fuel Partitioning" is provided. Highlights and issues of the conference are mentioned, as well as strategies for the future.

Effect of losartan on the Na+/Ca2+ exchanger in left ventricle of the insulin resistant and hypertensive hHTg rat.

As the Na+/Ca2+ exchanger plays an important role in the regulation of myocyte contractility, it has been suggested that alterations in this system might be involved in the development of insulin resistance and/or diabetes-induced myocardial alterations. Moreover, gene expression and function of the Na+/Ca2+ exchanger in states of combined hypertension and insulin resistance is of a special interest. Thus, we used hereditary hypertriglyceridemic (hHTg) rat (a model of genetically induced insulin resistance and hypertension) to study the effect of losartan, the blocker of type 1 angiotensin receptors, on the Na+/Ca2+ exchanger in the rat heart. We found that gene expression, but not activity of the Na+/Ca2+ exchanger was decreased in the left ventricle of hHTg rats when compared to their normotensive mates. No changes were observed in the right ventricle. In addition, losartan decreased mRNA levels of the Na+/Ca2+ exchanger in the left, but not in the right ventricle of normotensive rats. In hHTg rats, losartan had no effect on the gene expression of this transporter. Our results point to different modulatory pathways of Na+/Ca2+ exchanger in normotensive and hHTg rats.