Transmural differences in respiratory capacity across the rat left ventricle in health, aging, and streptozotocin-induced diabetes mellitus: evidence that mitochondrial dysfunction begins in the subepicardium.

In diabetic cardiomyopathy, ventricular dysfunction occurs in the absence of hypertension or atherosclerosis and is accompanied by altered myocardial substrate utilization and depressed mitochondrial respiration. It is not known if mitochondrial function differs across the left ventricular (LV) wall in diabetes. In the healthy heart, the inner subendocardial region demonstrates higher rates of blood flow, oxygen consumption, and ATP turnover compared with the outer subepicardial region, but published transmural respirometric measurements have not demonstrated differences. We aim to measure mitochondrial function in Wistar rat LV to determine the effects of age, streptozotocin-diabetes, and LV layer. High-resolution respirometry measured indexes of respiration in saponin-skinned fibers dissected from the LV subendocardium and subepicardium of 3-mo-old rats after 1 mo of streptozotocin-induced diabetes and 4-mo-old rats following 2 mo of diabetes. Heart rate and heartbeat duration were measured under isoflurane-anesthesia using a fetal-Doppler, and transmission electron microscopy was employed to observe ultrastructural differences. Heart rate decreased with age and diabetes, whereas heartbeat duration increased with diabetes. While there were no transmural respirational differences in young healthy rat hearts, both myocardial layers showed a respiratory depression with age (30-40%). In 1-mo diabetic rat hearts only subepicardial respiration was depressed, whereas after 2 mo diabetes, respiration in subendocardial and subepicardial layers was depressed and showed elevated leak (state 2) respiration. These data provide evidence that mitochondrial dysfunction is first detectable in the subepicardium of diabetic rat LV, whereas there are measureable changes in LV mitochondria after only 4 mo of aging.

Copper(II)-selective chelation improves function and antioxidant defences in cardiovascular tissues of rats as a model of diabetes: comparisons between triethylenetetramine and three less copper-selective transition-metal-targeted treatments.

AIMS/HYPOTHESIS: Treatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress. METHODS: Rats were made diabetic with streptozotocin (55 mg/kg, i. v.) and treated from 8 weeks after disease induction for the following 8 weeks with effective dosages of oral TETA, or one of three less Cu-selective transition-metal-targeted treatments: D-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine. RESULTS: Diabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevating the urinary albumin/creatinine ratio. Diabetes also lowered total antioxidant potential and heparan sulphate levels in cardiac and arterial tissues, and serum ferroxidase activity, whereas it elevated urinary heparan sulphate excretion. TETA treatment rectified or partially rectified all these defects, whereas the other three experimental treatments were ineffectual. By contrast, none of the four drug treatments lowered diabetes-mediated elevations of plasma glucose or lipid concentrations. CONCLUSIONS/INTERPRETATION: TETA may limit the cardiac and renal/vascular damage inflicted by diabetes through its actions to reinforce antioxidant defence mechanisms, probably acting through selective chelation of 'loosely-bound'/chelatable Cu(II). It may also improve heparan sulphate homeostasis and bolster antioxidant defence by increasing vascular extracellular superoxide dismutase activity. Urinary albumin/creatinine ratio might prove useful for monitoring TETA treatment.

A copper(II)-selective chelator ameliorates left-ventricular hypertrophy in type 2 diabetic patients: a randomised placebo-controlled study.

AIMS/HYPOTHESIS: Cu(II)-selective chelation with trientine ameliorates cardiovascular and renal disease in a model of diabetes in rats. Here, we tested the hypothesis that Cu(II)-selective chelation might improve left ventricular hypertrophy (LVH) in type 2 diabetic patients. METHODS: We performed a 12 month randomised placebo-controlled study of the effects of treatment with the Cu(II)-selective chelator trientine (triethylenetetramine dihydrochloride, 600 mg given orally twice daily) on LVH in diabetic patients (n = 15/group at baseline) in an outpatient setting wherein participants, caregivers and those assessing outcomes were blinded to group assignment. Using MRI, we measured left ventricular variables at baseline, and at months 6 and 12. The change from baseline in left ventricular mass indexed to body surface area (LVM(bsa)) was the primary endpoint variable. RESULTS: Diabetic patients had LVH with preserved ejection fraction at baseline. Trientine treatment decreased LVM(bsa) by 5.0 +/- 7.2 g/m(2) (mean +/- SD) at month 6 (when 14 trientine-treated and 14 placebo-treated participants were analysed; p = 0.0056 compared with placebo) and by 10.6 +/- 7.6 g/m(2) at month 12 (when nine trientine-treated and 13 placebo-treated participants were analysed; p = 0.0088), whereas LVM(bsa) was unchanged by placebo treatment. In a multiple-regression model that explained ~75% of variation (R (2) = 0.748, p = 0.001), cumulative urinary Cu excretion over 12 months was positively associated with trientine-evoked decreases in LVM(bsa). CONCLUSIONS/INTERPRETATION: Cu(II)-selective chelation merits further exploration as a potential pharmacotherapy for diabetic heart disease. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN 12609000053224 FUNDING: The Endocore Research Trust; Lottery Health New Zealand; the Maurice and Phyllis Paykel Trust; the Foundation of Research, Science and Technology (New Zealand); the Health Research Council of New Zealand; the Ministry of Education (New Zealand) through the Maurice Wilkins Centre for Molecular Biodiscovery; and the Protemix Corporation.

Coordination of mammary metabolism and blood flow after refeeding in rats.

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 mumol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.

Effect of high-fat meals and fatty acid saturation on postprandial levels of the hormones ghrelin and leptin in healthy men.

OBJECTIVE: Ghrelin and leptin play a role in control of food intake and adiposity but mechanisms regulating these hormones in man are poorly defined and evidence that dietary fats may have adverse effects is inconclusive. We investigated whether high-fat meals, which differed in saturated fatty acid (SFA) content acutely modified these hormones. DESIGN: Randomised, double-blind, crossover trial. A high-fat (HF) test meal (59 +/- 4 g fat; 71% of energy as fat) was given for breakfast on two occasions. Meals comprised either high (approximately 70:30) or low (approximately 55:45) saturated:unsaturated fatty acid (SFA:USFA) ratio. Fasting and postprandial measurements of serum total ghrelin (RIA), leptin (enzyme-linked immunosorbent assay (ELISA)) and insulin (RIA) were made over 6 h. Postprandial measurements were also made at 10 and 24 h following a fat-exclusion lunch, snack and dinner. SUBJECTS: A total of 18 lean, healthy men. RESULTS: There was no significant effect of the fatty meal (time, P > 0.05), nor a differential effect of SFA:USFA ratio (treatment*time, P > 0.05) on ghrelin over 6h. Leptin decreased in response to both HF treatments (time, P < 0.001) but increased SFA content did not further inhibit hormone secretion (treatment*time, P > 0.05). There was no significant correlation between ghrelin or leptin and circulating insulin (P>0.05). CONCLUSION: We conclude that HF diets may adversely effect serum leptin, although the circadian decrease may account in part for this response. Increasing dietary SFAs had no deleterious effects on leptin or total ghrelin.

Effect of moderate changes in dietary fatty acid profile on postprandial lipaemia, haemostatic and related CVD risk factors in healthy men.

OBJECTIVE: To investigate the effect of moderate changes in dietary fatty acid profile on postprandial risk factors for cardiovascular disease (CVD). DESIGN: Double-blind, randomised, crossover, intervention trial. SETTING: : University of Auckland Human Nutrition Unit, New Zealand. SUBJECTS: A total of 18 lean healthy men. INTERVENTION: A dairy butter fat modified to reduce the saturated:unsaturated fatty acid ratio and a conventional high saturated butter fat were given on two separate occasions as a high-fat test meal (59+/-4 g fat; 71 en% fat) at breakfast. A fat exclusion lunch, dinner and snacks were also given. Blood samples were collected at 0 (baseline), 1, 3, 6, 10 and 24 h. RESULTS: Maximum peak in total triacylglycerol (TAG) occurred 3 h postprandially and was highest on modified treatment (diet, P<0.05) due predominantly to increased TAG within the chylomicron-rich fraction. Transient peaks in total-, LDL- and HDL-cholesterol occurred postprandially, but did not differ between dietary treatments (P>0.05). There were no differential effects of diet on postprandial free fatty acids, apo A, apo B, glucose, insulin, amylin or haemostatic clotting factors (P>0.05). CONCLUSIONS: In a group of healthy young men, replacement of 16% of total saturated fatty acids by mono- and polyunsaturated fats within a dairy lipid did not induce postprandial changes in CVD risk that may be considered beneficial for health. SPONSORSHIP: Fonterra, Wellington; New Zealand.

Lipid-lowering effects of a modified butter-fat: a controlled intervention trial in healthy men.

OBJECTIVE: To investigate the lipid-lowering potential of a butter-fat modified through manipulations in bovine feeding to increase the unsaturated:saturated fatty acid ratio. DESIGN: Double-blind, randomised, cross-over intervention trial. SETTING: University of Auckland Human Nutrition Unit, New Zealand. SUBJECTS: Twenty healthy, male subjects. INTERVENTION: A residential trial in which all foods and beverages were provided during two intervention periods, comprising 3 weeks of high unsaturated 'modified' vs. 3 weeks of saturated 'control' butter feeding separated by a 4 week washout. Diets were of typical composition of 39 percentage energy (en%) fat (20 en% butter-fat), 48 en% CHO, 13 en% protein. RESULTS: There was a significant decrease in both total (P<0.05, -7.9%) and LDL-cholesterol (P<0.01, -9.5%) during modified butter feeding. There was no significant effect of treatment on a range of other risk factors including HDL-cholesterol, triglyceride, apolipoprotein A or B, nonesterified fatty acids (NEFA), haemostatic clotting factor VII and fibrinogen or glucose (P>0.05). Subjects were maintained in energy balance and there was no significant change in body weight during intervention. Butter-fat composition alone differed between treatments. CONCLUSIONS: A significant improvement in cardiovascular risk can be achieved by moderate changes in dietary fatty acid profile, achieved through a common and well accepted food source, butter-fat.

Site-specific cross-linking of collagen peptides by lysyl advanced glycation endproducts.

Cross-linking of proteins by advanced glycation endproducts (AGEs) causes a host of pathological conditions but their exact roles are unknown. Cross-linking lysyl AGEs were synthesized and incorporated into two types of collagen peptides. The utility of these cross-linked peptides for biochemical investigations was demonstrated by proteolysis studies and circular dichroism.

Selective divalent copper chelation for the treatment of diabetes mellitus.

Oxidative stress and mitochondrial dysfunction have been identified by many workers as key pathogenic mechanisms in ageing-related metabolic, cardiovascular and neurodegenerative diseases (for example diabetes mellitus, heart failure and Alzheimer's disease). However, although numerous molecular mechanisms have been advanced to account for these processes, their precise nature remains obscure. This author has previously suggested that, in such diseases, these two mechanisms are likely to occur as manifestations of a single underlying disturbance of copper regulation. Copper is an essential but highly-toxic trace metal that is closely regulated in biological systems. Several rare genetic disorders of copper homeostasis are known in humans: these primarily affect various proteins that mediate intracellular copper transport processes, and can lead either to tissue copper deficiency or overload states. These examples illustrate how impaired regulation of copper transport pathways can cause organ damage and provide important insights into the impact of defects in specific molecular processes, including those catalyzed by the copper-transporting ATPases, ATP7A (mutated in Menkes disease), ATP7B (Wilson's disease), and the copper chaperones such as those for cytochrome c oxidase, SCO1 and SCO2. In diabetes, impaired copper regulation manifests as elevations in urinary CuII excretion, systemic chelatable-CuII and full copper balance, in increased pro-oxidant stress and defective antioxidant defenses, and in progressive damage to the blood vessels, heart, kidneys, retina and nerves. Linkages between dysregulated copper and organ damage can be demonstrated by CuII-selective chelation, which simultaneously prevents/reverses both copper dysregulation and organ damage. Pathogenic structures in blood vessels that contribute to binding and localization of catalytically-active CuII probably include advanced glycation end products (AGEs), as well as atherosclerotic plaque: the latter probably undergoes AGE-modification itself. Defective copper regulation mediates organ damage through two general processes that occur simultaneously in the same individual: elevation of CuII-mediated pro-oxidant stress and impairment of copper-catalyzed antioxidant defence mechanisms. This author has proposed that diabetes-evoked copper dysregulation is an important new target for therapeutic intervention to prevent/reverse organ damage in diabetes, heart failure, and neurodegenerative diseases, and that triethylenetetramine (TETA) is the first in a new class of anti-diabetic molecules, which function by targetting these copper-mediated pathogenic mechanisms. TETA prevents tissue damage and causes organ regeneration by acting as a highly-selective CuII chelator which suppresses copper-mediated oxidative stress and restores anti-oxidant defenses. My group has employed TETA in a comprehensive programme of nonclinical studies and proof-of-principle clinical trials, thereby characterizing copper dysregulation in diabetes and identifying numerous linked cellular and molecular mechanisms though which TETA exerts its therapeutic actions. Many of the results obtained in nonclinical models with respect to the molecular mechanisms of diabetic organ damage have not yet been replicated in patients' tissues so their applicability to the human disease must be considered as inferential until the results of informative clinical studies become available. Based on evidence from the studies reviewed herein, trientine is now proceeding into the later stages of pharmaceutical development for the treatment of heart failure and other diabetic complications.

A copper(II)-selective chelator ameliorates diabetes-evoked renal fibrosis and albuminuria, and suppresses pathogenic TGF-beta activation in the kidneys of rats used as a model of diabetes.

AIMS/HYPOTHESIS: The selective Cu(II) chelator triethylenetetramine (TETA) extracts systemic Cu(II) into the urine of diabetic humans and rats as a model of diabetes, and in the process also normalises hallmarks of diabetic heart disease. However, the role of Cu and its response to TETA in animals with diabetic nephropathy were previously unknown. Here, we report the effects of TETA treatment on Cu and other essential elements, as well as on indices of renal injury and known pathogenic molecular processes, in kidneys from a rat model of diabetes. METHODS: Rats at 8 weeks after streptozotocin-induction of diabetes were treated with oral TETA (34 mg/day in drinking water) for a further 8 weeks and then compared with untreated diabetic control animals. RESULTS: Renal tissue Cu was substantively elevated by diabetes and normalised by TETA, which also suppressed whole-kidney and glomerular hypertrophy without lowering blood glucose. The urinary albumin: creatinine ratio was significantly elevated in the rat model of diabetes but lowered by TETA. Total collagen was also elevated in diabetic kidneys and significantly improved by TETA. Furthermore, renal cortex levels of TGF-beta1, MAD homologue (SMAD) 4, phosphorylated SMAD2, fibronectin-1, collagen-III, collagen-IV, plasminogen activator inhibitor-1 and semicarbazide-sensitive amine oxidase all tended to be elevated in diabetes and normalised by TETA. CONCLUSIONS/INTERPRETATION: Dysregulation of renal Cu homeostasis may be a key event eliciting development of diabetic nephropathy. Selective Cu(II) chelation can protect against pathogenic mechanisms that lead to or cause diabetic nephropathy and might be clinically useful in the treatment of early-stage diabetic kidney disease.

Preptin, another peptide product of the pancreatic beta-cell, is osteogenic in vitro and in vivo.

Several hormones that regulate nutritional status also impact on bone metabolism. Preptin is a recently isolated 34-amino acid peptide hormone that is cosecreted with insulin and amylin from the pancreatic beta-cells. Preptin corresponds to Asp(69)-Leu(102) of pro-IGF-II. Increased circulating levels of a pro-IGF-II peptide complexed with IGF-binding protein-2 have been implicated in the high bone mass phenotype observed in patients with chronic hepatitis C infection. We have assessed preptin's activities on bone. Preptin dose-dependently stimulated the proliferation (cell number and DNA synthesis) of primary fetal rat osteoblasts and osteoblast-like cell lines at periphysiological concentrations (>10(-11) M). In addition, thymidine incorporation was stimulated in murine neonatal calvarial organ culture, likely reflecting the proliferation of cells from the osteoblast lineage. Preptin did not affect bone resorption in this model. Preptin induced phosphorylation of p42/p44 MAP kinases in osteoblastic cells in a dose-dependent manner (10(-8)-10(-10) M), and its proliferative effects on primary osteoblasts were blocked by MAP kinase kinase inhibitors. Preptin also reduced osteoblast apoptosis induced by serum deprivation, reducing the number of apoptotic cells by >20%. In vivo administration of preptin increased bone area and mineralizing surface in adult mice. These data demonstrate that preptin, which is cosecreted from the pancreatic beta-cell with amylin and insulin, is anabolic to bone and may contribute to the preservation of bone mass observed in hyperinsulinemic states such as obesity.

Amylin gene promoter mutations predispose to Type 2 diabetes in New Zealand Maori.

AIMS/HYPOTHESIS: Amylin gene mutations are known to predispose Chinese and Japanese subjects, but not Caucasian subjects, to Type 2 diabetes. New Zealand Maori, who have a high prevalence of Type 2 diabetes, have genetic origins in South East Asia. Amylin gene mutations could therefore predispose New Zealand Maori to Type 2 diabetes. METHODS: The amylin gene was screened for mutations in the proximal promoter region, exons 1 and 2, intron 1, and coding region of exon 3 by polymerase chain reaction amplification and direct sequencing of 131 Type 2 diabetic Maori patients and 258 non-diabetic Maori control subjects. RESULTS: We identified three new amylin gene mutations: two mutations in the promoter region (-215T>G and -132G>A) and a missense mutation in exon 3 (Q10R). The -215T>G mutation was observed in 5.4% of Type 2 Maori diabetic patients and predisposed the carrier to diabetes with a relative risk of 7.23. The -215T>G mutation was inherited with a previously described amylin promoter polymorphism (-230A>C) in 3% of the Maori with Type 2 diabetes, which suggests linkage disequilibrium exists between these two mutations. The -230A>C polymorphism on its own, however, was not associated with Type 2 diabetes in Maori subjects. The -132G>A and Q10R mutations were both observed in 0.76% of Type 2 diabetic patients and were absent in non-diabetic subjects. CONCLUSION/INTERPRETATION: The amylin gene mutations identified in this study are associated with Type 2 diabetes in 7% of Maori. Amylin is likely to be an important susceptibility gene for Type 2 diabetes in Maori people.