Malic enzyme is present in mouse islets and modulates insulin secretion.

AIMS/HYPOTHESIS: The pyruvate-malate shuttle is a metabolic cycle in pancreatic beta cells and is important for beta cell function. Cytosolic malic enzyme (ME) carries out an essential step in the shuttle by converting malate to pyruvate and generating NADPH. In rat islets the pyruvate-malate shuttle may regulate insulin secretion and it has been shown to play a critical role in adaptation to obesity and insulin resistance. However, ME has not been demonstrated in mouse islets and three reports indicate that mouse islets contain no ME activity. If mouse islets lack ME, rat and mouse islets must regulate insulin secretion by different mechanisms. METHODS: We measured ME activity by a fluorometric enzymatic assay and Me mRNA by real-time PCR. ME activity was also measured in streptozotocin-treated mouse islets. FACS-purified beta cells were obtained from MIP-GFP mouse islets, agouti-L obese mouse islets and mouse beta cell line MIN-6. Insulin secretion and NADPH/NADP(+) ratios were measured in Me siRNA-treated beta cells. RESULTS: ME activity and Me mRNA were present in C57BL/6 mouse islets. ME activity was reduced in streptozotocin-treated mouse islets. ME activity was also measurable in FACS-purified mouse beta cells. In addition, ME activity was significantly increased in obese agouti-L mouse islets and the mouse MIN-6 cell line. Me siRNA inhibited ME activity and reduced glucose-stimulated insulin secretion and also inhibited NADPH products. CONCLUSIONS/INTERPRETATION: Mouse islets contain ME, which plays a significant role in regulating insulin secretion.

Functional changes in baroreceptor afferent, central and efferent components of the baroreflex circuitry in type 1 diabetic mice (OVE26).

Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes mellitus induced functional changes of neural components at multiple sites within the baroreflex arc. Type 1 diabetic mice (OVE26) and FVB control mice were anesthetized. Baroreflex-mediated HR responses to sodium nitroprusside- (SNP) and phenylephrine- (PE) induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor function was characterized by measuring the percent (%) change of baseline integrated aortic depressor nerve activity (Int ADNA) in response to SNP- and PE-induced MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve (ADN) and the right vagus nerve were assessed. Compared with FVB control mice, we found in OVE26 mice that (1) baroreflex-mediated bradycardia and tachycardia were significantly reduced. (2) The baroreceptor afferent function in response to MAP increase did not differ, as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was significantly attenuated. (3) The maximum amplitude of bradycardic responses to right vagal efferent stimulation was augmented. (4) In contrast, the maximum amplitude of bradycardic responses to left ADN stimulation was decreased. Since Int ADNA was preserved in response to MAP increase and HR responses to vagal efferent stimulation were augmented, we conclude that a deficit of the central mediation of baroreflex HR contributes to the overall attenuation of baroreflex sensitivity in OVE26 mice. The successful conduction of physiological experiments on the ADN in OVE26 mice may provide a foundation for the understanding of cellular and molecular mechanisms of diabetes-induced cardiac neuropathy.

Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity.

Metallothionein (MT) may provide protection against doxorubicin-induced heart damage. To test this hypothesis, a heart-specific promoter was used to drive the expression of human MT-IIa gene in transgenic mice. Four healthy transgenic mouse lines were produced. Cardiac MT was constitutively overexpressed from 10- to 130-fold higher than normal. The MT concentration was not altered in liver, kidneys, lungs, or skeletal muscles. Other antioxidant components including glutathione, glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase were not altered in the MT-overexpressing heart. Mice (7-wk-old) from transgenic lines expressing MT activity 10- or 130-fold higher than normal and from nontransgenic controls were treated intraperitoneally with doxorubicin at a single dose of 20 mg/kg, and were killed on the 4th day after treatment. As compared to normal controls, transgenic mice exhibited a significant resistance to in vivo doxorubicin-induced cardiac morphological changes, and the increase in serum creatine phosphokinase activity. Atria isolated from transgenic mice and treated with doxorubicin in tissue bath was also more resistant to functional damage induced by this drug. The results provide direct evidence for the role of MT in cardioprotection against doxorubicin toxicity.

Morphology and topography of nucleus ambiguus projections to cardiac ganglia in rats and mice.

Vagal efferent axons from the nucleus ambiguus (NA) innervate ganglionated plexuses in the dorsal surface of cardiac atria, which in turn, may have different functional roles in cardiac regulation. However, the morphology and topography of vagal efferent projections to these ganglionated plexuses in rats and mice have not been well delineated. In the present study, we injected the tracer 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine methanesulfonate (DiI) into the left NA to label vagal efferent axons and terminals in cardiac ganglia and administered Fluoro-Gold (FG) i.p. to stain cardiac ganglia. Then, we used confocal microscopy and a Neurolucida 3-D Digitization System to qualitatively and quantitatively examine the distribution and structure of cardiac ganglia, and NA efferent projections to cardiac ganglia in the whole-mounts of Sprague-Dawley (SD) rats and FVB mice. Our observations were: 1) Cardiac ganglia of different shapes and sizes were distributed in the sinoatrial (SA) node, atrioventricular (AV) node, and lower pulmonary vein (LPV) regions on the dorsal surface of the atria. In each region, several ganglia formed a ganglionated plexus. The plexuses at different locations were interconnected by nerves. 2) Vagal efferent fibers ramified within cardiac ganglia, formed a complex network of axons, and innervated cardiac ganglia with very dense basket endings around individual cardiac principal neurons (PNs). 3) The percent of the PNs in cardiac ganglia which were innervated by DiI-labeled axons was 54.3+/-3.2% in mice vs. 53.2+/-3.2% in rats (P>0.10). 4) The density of axonal putative-synaptic varicosities on the surface of PNs was 0.15+/-0.02/microm(2) in mice vs. 0.16+/-0.02/microm(2) in rats (P>0.10). Thus, the distributions of cardiac ganglia and vagal efferent projections to cardiac ganglia in mice and rats were quite similar both qualitatively and quantitatively. Our study provides the structural foundation for future investigation of functional differentiation of ganglionated plexuses and the brain-heart circuitry in rodent models of human disease.

Impaired Albumin Uptake and Processing Promote Albuminuria in OVE26 Diabetic Mice.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes.

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced beta-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD+. Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect beta-cell DNA and thereby prevent STZ-induced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in beta-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD+. To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced beta-cell death.

Chicken calmodulin promoter activity in proliferating and differentiated cells.

A 1218-base pair (bp) portion of the chicken calmodulin promoter was sequenced and assayed for promoter activity. This portion of the promoter was found sufficient to produce accurate transcriptional initiation. The promoter sequence was GC rich, particularly in the 700 bp region 5' to the cap site. Eight plasmids were prepared containing the first calmodulin exon and 30-1218 bp of the promoter, ligated to the reporter gene chloramphenicol acetyl transferase. In chicken embryonic fibroblasts and proliferating BC3H-1 cells promoter activity increased progressively with increasing promoter length up to 617 bp. Extension of the promoter beyond 617 bp inhibited expression, as did sequences within the first calmodulin exon. In BC3H-1 cells differentiation was found to reduce calmodulin mRNA levels approximately 3-fold. Activity of the calmodulin promoter constructs also decreased by a similar extent with differentiation. Sequences up to 234 bp 5' to the calmodulin cap site were markedly less effective in elevating chloramphenicol acetyl transferase activity in differentiated BC3H-1 cells than in proliferating cells and may account for the lower overall activity of the calmodulin promoter in these cells. Within this region several sequences were identified, including an extensive homology to the rat calmodulin I gene promoter that could be significant in regulation of calmodulin expression.

Targeted overexpression of an inactive calmodulin that binds Ca2+ to the mouse pancreatic beta-cell results in impaired secretion and chronic hyperglycemia.

We have previously reported that elevated levels of calmodulin in pancreatic beta-cells of mice resulted in a unique secretory defect. To determine if this effect was due to Ca2+ buffering, a mutant form of calmodulin that has an eight-amino acid deletion in the central helix (CaM-8) was used. The mutated calmodulin binds Ca2+ normally, but alters the ability to interact with known Ca2+/calmodulin-activated enzymes. In vitro competition analysis using HIT cell extracts verified that in the presence of Ca2+, CaM-8 exhibited at least a 100-fold lower affinity for calmodulin-binding proteins than did normal CaM in this model beta-cell. Transgenic mice were then generated by targeting the CaM-8 to pancreatic beta-cells. The CaM-8 mice were normoglycemic at birth, but developed a hyperglycemic condition starting at about 6 days of age. This condition was progressive and characterized by elevated blood glucose that coincided with reduced levels of pancreatic insulin and low circulating serum insulin levels. Hormone measurements and immunohistochemical analysis revealed that islets exhibited a nonimmune reduction of insulin immunoreactive beta-cells, reduced amounts of insulin, and a 5-fold higher level of CaM-8 protein relative to normal CaM protein. Perifusion assays were used to test the secretion response to glucose. CaM-8 islets demonstrated a reduction in first and second phase insulin secretion, which became progressively worse with age. Depolarization of the membrane with 50 mM K+ in the presence of high glucose did not significantly improve secretion. Carbachol, which is thought to act in beta-cells through the release of intracellular Ca2+ stores and activation of protein kinase-C, restored both phases of secretion to normal levels. These results suggest that disruption of intracellular Ca2+ homeostasis alone is sufficient to interfere with the insulin secretion pathway.

Elevated beta-cell calmodulin produces a unique insulin secretory defect in transgenic mice.

Transgenic mice with elevated levels of beta-cell calmodulin develop severe diabetes even though pancreatic beta-cells contain reserve levels of insulin. Electron microscopic examination of transgenic pancreas confirmed the presence of abundant insulin secretory granules and failed to reveal obvious morphological abnormalities. These observations suggested that excess calmodulin may specifically impair the secretory process. To directly assess the effect of excess calmodulin on beta-cell function we have isolated pancreatic islets from transgenic animals. Transgenic islets from 6- to 8-day-old mice used 40% less glucose than normal islets and contained 58% of the normal insulin content, 90% of the normal glucagon content, and 5-fold higher levels of calmodulin than islets from control mice of the same age. Parallel perifusions of normal and transgenic islets confirmed that excess calmodulin inhibited glucose-stimulated insulin secretion; first phase secretion was reduced by 60%, and second phase secretion was essentially absent. Static assays were performed to assess the response to other secretagogues. All fuel secretagogues tested were ineffective in stimulating insulin secretion from transgenic islets. Secretion in response to depolarizing levels of potassium was also severely impaired. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine increased transgenic secretion, but not to the level obtained in normal islets. Of the compounds examined, only phorbol 12-myristate 13-acetate and carbachol, two substances thought to act in beta-cells by stimulation of protein kinase-C, produced equivalent secretion in normal and transgenic islets. Phorbol 12-myristate 13-acetate also appeared to restore second phase secretion in transgenic islets. These results indicate that the initial period of calmodulin-induced diabetes is due to a secretory defect. This defect appears to be distal to membrane depolarization and is selective for the second phase of insulin secretion.

Overexpression of catalase provides partial protection to transgenic mouse beta cells.

Pancreatic beta cells are sensitive to reactive oxygen species and this may play an important role in type 1 diabetes and during transplantation. Beta cells contain low levels of enzyme systems that protect against reactive oxygen species. The weakest link in their protection system is a deficiency in the ability to detoxify hydrogen peroxide by the enzymes glutathione peroxidase and catalase. We hypothesize that the deficit in the ability to dispose of reactive oxygen species is responsible for the unusual sensitivity of beta cells and that increasing protection will result in more resistant beta cells. To test these hypotheses we have produced transgenic mice with increased beta cell levels of catalase. Seven lines of catalase transgenic mice were produced using the insulin promoter to direct pancreatic beta cell specific expression. Catalase activity in islets from these mice was increased by as much as 50-fold. Northern blot analysis of several tissues indicated that overexpression was specific to the pancreatic islet. Catalase overexpression had no detrimental effects on islet function. To test whether increased catalase activity could protect the transgenic islets we exposed them to hydrogen peroxide, streptozocin, and interleukin-1beta. Fifty-fold overexpression of catalase produced marked protection of islet insulin secretion against hydrogen peroxide and significantly reduced the diabetogenic effect of streptozocin in vivo. However, catalase overexpression did not provide protection against interleukin-1beta toxicity and did not alter the effects of syngeneic and allogenic transplantation on islet insulin content. Our results indicate that in the pancreatic beta cell overexpression of catalase is protective against some beta cell toxins and is compatible with normal function.

Quantitative immunocytochemical study of islet cell populations in diabetic calmodulin-transgenic mice.

The present study describes the changes in the endocrine pancreas of severely diabetic calmodulin-transgenic mice using light microscopic immunocytochemical and morphometric techniques. A marked reduction in the number and volume of islets, together with distortion of their normal architecture, was found in diabetic mice. In addition, the volume density of both endocrine tissue and B-cells was decreased. An irregular distribution of non-B-cells was also observed in diabetic animals. The volume density and the percentage of A-cells appeared increased. However, when quantified per area unit, the number of all the islet cell types diminished, although only the decrease in B-cell number was statistically significant. The decrease in B-cell mass might account for the diabetic state developed in this animal model.

Prenatal ethanol exposure alters ventricular myocyte contractile function in the offspring of rats: influence of maternal Mg2+ supplementation.

Fetal alcohol syndrome (FAS) is often associated with cardiac hypertrophy and impaired ventricular function in a manner similar to postnatal chronic alcohol ingestion. Chronic alcoholism has been shown to lead to hypomagnesemia, and dietary Mg2+ supplementation was shown to ameliorate ethanol- induced cardiovascular dysfunction such as hypertension. However, the role of gestational Mg2+ supplementation on FAS-related cardiac dysfunction is unknown. This study was conducted to examine the influence of gestational dietary Mg2+ supplementation on prenatal ethanol exposure-induced cardiac contractile response at the ventricular myocyte level. Timed-pregnancy female rats were fed from gestation day 2 with liquid diets containing 0.13 g/L Mg2+ supplemented with ethanol (36%) or additional Mg2+ (0.52 g/L), or both. The pups were maintained on standard rat chow through adulthood, and ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an IonOptix soft-edge system, and intracellular Ca2+ transients were measured as changes in fura-2 fluorescence intensity (Delta FFI). Offspring from all groups displayed similar growth curves. Myocytes from the ethanol group exhibited reduced cell length, enhanced peak shortening (PS), and shortened time to 90% relengthening (TR90) associated with a normal Delta FFI and time to PS (TPS). Mg2+ reverted the prenatal ethanol-induced alteration in PS and maximal velocity of relengthening. However, it shortened TPS and TR90, and altered the Delta FFI, as well as Ca2+ decay rate by itself. Additionally, myocytes from the ethanol group exhibited impaired responsiveness to increased extracellular Ca2+ or stimulating frequency, which were restored by gestational Mg2+ supplementation. These data suggest that although gestational Mg2+ supplementation may be beneficial to certain cardiac contractile dysfunctions in offspring of alcoholic mothers, caution must be taken, as Mg2+ supplementation affects cell mechanics itself.

Altered response to apomorphine and haloperidol after nine days of cocaine injections.

Sprague Dawley rats, pretreated with nine daily injections of 20 mg/Kg cocaine or saline, were evaluated for aspects of their behavioral response to apomorphine, haloperidol, or cocaine, twenty-four hours after their last pretreatment injection. Data obtained from saline and cocaine pretreated animals indicated that: cocaine pretreated rats were more sensitive to haloperidol-induced catalepsy, less responsive to some of the stereotypic effects of apomorphine and similar in their responses to the anticataleptic properties of cocaine.

[Structure and ultrastructure of the endocrine pancreas in diabetic transgenic mice]. / Estructura y ultraestructura del páncreas endocrino de ratones transgénicos diabéticos.

The aim of the present study was to confirm the structural changes and to establish the ultrastructural alterations that occur in the endocrine pancreas of mice with an induced insulin-dependent diabetes mellitus (IDDM) syndrome. For that purpose, we used transgenic mice (OVE 27) that overexpress a calmodulin gene in the beta cells of the endocrine pancreas. In these animals, the excess of calmodulin decreases the cytosolic calcium levels in beta cells, leading to morphological and functional alterations that produce a severe IDDM. Sections of pancreas (tail) from 4 male 5-week-old diabetic mice (glycemia: 376 +/- 2 mg/dl) and from 4 normal age-matched males (glycemia: 113 +/- 13 mg/dl) were processed. Light microscopic immunohistochemical observations confirmed a decrease in the number and size of pancreatic islets in transgenic mice, together with a disruption in their architecture, without an associated inflammatory response. The ultrastructural studies revealed diverse degrees of injury in the beta cells, such as the presence of membrane interdigitations and alterations in their organelles and secretory granules. These findings are in agreement with the quantitative and functional impairment of beta cells, coexisting with a normal appearance of non-beta cell populations within the pancreatic islets. Our results demonstrate the existence of ultrastructural changes in the pancreatic beta cells of the experimental model studied. Such changes, together with the immunohistochemical alterations previously described, contribute to explain the appearance of a diabetic syndrome in these animals.

Impaired baroreflex control of renal sympathetic nerve activity in type 1 diabetic mice (OVE26).

To investigate the effects of chronic diabetes on baroreflex control of renal sympathetic nerve activity (RSNA), OVE26 diabetic (transgenic mouse line which develops hyperglycemia within the first 3 weeks after birth) and FVB control mice 5-6 months old were studied. Under anesthesia, RSNA in response to sodium nitroprusside (SNP)- and phenylephrine (PE)-induced mean arterial pressure changes (DeltaMAP) were measured. Baroreflex-induced inhibition of RSNA during PE infusion was characterized using the sigmoid logistic function curve. Baroreflex-induced excitation of RSNA during SNP infusion was characterized by the RSNA vs. DeltaMAP relationship. Mean arterial pressure (MAP) responses to the left aortic depressor nerve (ADN) stimulation were evaluated. Compared to FVB control, we found in OVE26 mice that (1) RSNA in response to MAP increase during PE infusion was dramatically reduced, as characterized by the maximal gain of the RSNA sigmoid logistic function curve (FVB: -20.0+/-5.1; OVE26: -7.6+/-0.8%/mm Hg, P<0.05); (2) RSNA in response to MAP decrease during SNP infusion was also attenuated (P<0.05); (3) MAP responses to ADN stimulation were reduced (P<0.05). We concluded that chronic diabetes impairs baroreflex control of RSNA in OVE26 diabetic mice. The use of the transgenic OVE26 diabetic mouse model may underlie a foundation for the further understanding of diabetes-induced autonomic neuropathy.

The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells.

AIMS/HYPOTHESIS: Pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure. METHODS: To confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells. RESULTS: Treatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP(+) ratio and activity of the pyruvate-malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion. CONCLUSIONS/INTERPRETATION: Our findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation.

Enhanced rat beta-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway.

Islet beta-cell proliferation is a very important component of beta-cell adaptation to insulin resistance and prevention of type 2 diabetes mellitus. However, we know little about the mechanisms of beta-cell proliferation. We now investigate the relationship between pyruvate carboxylase (PC) pathway activity and islet cell proliferation 5 days after 60% pancreatectomy (Px). Islet cell number, protein, and DNA content, indicators of beta-cell proliferation, were increased two- to threefold 5 days after Px. PC and pyruvate dehydrogenase (PDH) activities increased only approximately 1.3-fold; however, islet pyruvate content and malate release from isolated islet mitochondria were approximately threefold increased in Px islets. The latter is an indicator of pyruvate-malate cycle activity, indicating that most of the increased pyruvate was converted to oxaloacetate (OAA) through the PC pathway. The contents of OAA and malate, intermediates of the pyruvate-malate cycle, were also increased threefold. PDH and citrate content were only slightly increased. Importantly, the changes in cell proliferation parameters, glucose utilization, and oxidation and malate release were partially blocked by in vivo treatment with the PC inhibitor phenylacetic acid. Our results suggest that enhanced PC pathway in Px islets may have an important role in islet cell proliferation.

Long-term effect of maternal obesity on pancreatic beta cells of offspring: reduced beta cell adaptation to high glucose and high-fat diet challenges in adult female mouse offspring.

AIM/HYPOTHESIS: Obesity is a global problem with high risks of cardiovascular diseases, stroke and type 2 diabetes. It is well known that maternal obesity affects offspring by inducing malformation, functional abnormalities in many organs and cells, and by increased risk of obesity and type 2 diabetes. However, little is known about abnormalities induced by maternal obesity in pancreatic beta cells of offspring. METHODS: We used mouse mothers with the Agouti yellow modification on a C57BL/6 background as a maternal model of normoglycaemic obesity, and produced Agouti-negative offspring. Half of the offspring were fed a high-fat diet. Offspring glucose tolerance was tested at different ages, and animals were killed at 50 weeks of age for islet function analysis. RESULTS: Maternal obesity impaired glucose tolerance in female offspring fed a high-fat diet, and significantly reduced insulin secretion at 50 weeks of age in female offspring that had been fed a normal diet and high-fat diet. Insulin secretion and glucose potentiation from these islets were significantly reduced. Islet protein, DNA and insulin contents were increased while glyceraldehyde-3-phosphate dehydrogenase and transketolase activities were reduced in female offspring. CONCLUSIONS/INTERPRETATION: Our results indicate that maternal obesity has a long-term effect on the beta cells of female, but not of male, offspring, and leads to increased risk of gestational diabetes and type 2 diabetes in the offspring's later lives.

Metallothionein alleviates cardiac contractile dysfunction induced by insulin resistance: role of Akt phosphorylation, PTB1B, PPARgamma and c-Jun.

AIMS/HYPOTHESIS: Insulin resistance is concomitant with metabolic syndrome, oxidative stress and cardiac contractile dysfunction. However, the causal relationship between oxidative stress and cardiac dysfunction is unknown. This study was designed to determine the impact of overexpression of the cardiac antioxidant metallothionein on cardiac dysfunction induced by insulin resistance in mice. METHODS: Whole-body insulin resistance was generated in wild-type FVB and metallothionein transgenic mice by feeding them with sucrose for 12 weeks. Contractile and intracellular Ca(2+) properties were evaluated in ventricular myocytes using an IonOptix system. The contractile indices analysed included: peak shortening (PS), time to 90% PS (TPS(90)), time to 90% relengthening (TR(90)), half-width duration, maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt), fura-fluorescence intensity change (DeltaFFI) and decay rate (tau). RESULTS: The sucrose-fed mice displayed glucose intolerance, enhanced oxidative stress, hyperinsulinaemia, hypertriglyceridaemia and normal body weight. Compared with myocytes in starch-fed mice, those from sucrose-fed mice exhibited depressed PS, +dL/dt, -dL/dt, prolonged TR(90) and decay rate, and reduced DeltaFFI associated with normal TPS(90) and half-width duration. Western blot analysis revealed enhanced basal, but blunted insulin (15 mU/g)-stimulated Akt phosphorylation. It also showed elevated expression of insulin receptor beta, insulin receptor tyrosine phosphorylation, peroxisome proliferator-activated receptor gamma, protein tyrosine phosphatase 1B and phosphorylation of the transcription factor c-Jun, associated with a reduced fold increase of insulin-stimulated insulin receptor tyrosine phosphorylation in sucrose-fed mice. All western blot findings may be attenuated or ablated by metallothionein. CONCLUSIONS/INTERPRETATION: These data indicate that oxidative stress may play an important role in cardiac contractile dysfunction associated with glucose intolerance and possibly related to alteration in insulin signalling at the receptor and post-receptor levels.

Changes in the effects of cocaine during chronic treatment.

The principle object of the study was to identify the changes in the rat's response to cocaine which occurred during chronic administration of the drug. Doses of 20 and 35 mg/Kg cocaine were administered at intervals of 12 and 24 hours, for 300 days or until their effects proved fatal. Four main effects were observed: sensitization to cocaine's convulsant and behavioral activating properties, tolerance to its weight loss effects and decreased response duration. Convulsant sensitization occurred primarily among rats receiving the higher dose of cocaine. All other effects occurred with equal frequency in rats at both doses. None of the changes were dependent on the interval between injections.