Biomedical subjects

P N Epstein

Publications and source records attributed to P N Epstein.

At least 19 recordsLinked to original sources

PubMed2005

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.

Animals↗

PubMed2005

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.

Acclimatization↗

PubMed2004

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.

Animals↗

PubMed2001

Characterization of contractile function in diabetic hypertensive cardiomyopathy in adult rat ventricular myocytes.

Diabetes and hypertension both produce myocardial dysfunction that accelerates cardiovascular morbidity and mortality. Coexistence of the two often results in a more severe cardiomyopathy than either process alone. The purpose of this study was to characterize the contractile function of diabetic hypertensive cardiomyopathy at the single myocyte level. Adult spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were made diabetic with a single injection (55 mg/kg) of streptozotocin (STZ). Contractile properties of ventricular myocytes were evaluated, including peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90) and maximal velocities of shortening/relengthening (+/-dL/d t). The experimental animals exhibited enlarged heart size, elevated blood glucose and systolic blood pressure. PS was unchanged (SHR), enhanced (WKY-STZ) or depressed (SHR-STZ) compared to control (WKY). Myocytes from all experimental groups displayed prolonged TPS and TR90 compared to the WKY group, although only those from the hypertensive groups (SHR, SHR-STZ) were associated with reduced +/-dL/d t. Additionally, myocytes from the WKY-STZ but not the SHR or the SHR-STZ groups exhibited impaired responsiveness to increased extracellular Ca2+. Myocytes from the SHR-STZ group displayed a leftward shift of the stimulus frequency-peak shortening response curve compared to the WKY group. These results confirmed observations at the multicellular levels that combination of diabetes and hypertension results in a greater impairment of cardiac contractile function than is seen with either disease alone.

Animals↗

PubMed2001

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.

Aging↗

PubMed2001

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.

Animals↗

PubMed2000

Diabetes enhances acetaldehyde-induced depression of cardiac myocyte contraction.

It is well established that cardiomyopathy is a consistent feature of diabetes and that alcohol consumption increases the risk of cardiovascular disease among diabetic subjects. Acetaldehyde (ACA), the main ethanol metabolite, is considered to play a role in the ethanol-induced cardiac dysfunction. It has been reported recently that the negative inotropic effect of ACA was more potent in the diabetic myocardium. To determine whether the disparate ACA-induced myocardial depression in diabetes is due to intrinsic alterations at the cellular level, mechanical properties in response to ACA were evaluated in ventricular myocytes from both normal and streptozotocin-induced diabetic rat hearts. Myocytes were electrically stimulated to contract at 0.5 Hz and contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and maximal velocities of shortening and relengthening (+/-dL/dt). Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes. ACA (0. 1-30 mM) disproportionately depressed PS in a dose-dependent manner, in myocytes from diabetic hearts compared to normal hearts. Interestingly, the degree of inhibition in DeltaFFI was similar in both groups. Neither the duration nor maximal velocities of shortening and relengthening were affected by ACA in either group. These results are the first to suggest that enhanced ACA-induced myocardial depression in diabetes is due to disparate intrinsic actions on individual myocytes. The mechanism underlying the alteration of ACA-induced myocardial depression may be due, in part, to depressed Ca(2+) responsiveness in diabetic hearts.

Acetaldehyde↗

PubMed1999

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.

Animals↗

PubMed1999

Prostate-specific human N-acetyltransferase 2 (NAT2) expression in the mouse.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine identified in the human diet and in cigarette smoke that produces prostate tumors in the rat. PhIP is bioactivated by cytochrome P-450 enzymes to N-hydroxylated metabolites that undergo further activation by conjugation enzymes, including the N-acetyltransferases, NAT1 and NAT2. To investigate the role of prostate-specific expression of human N-acetyltransferase 2 (NAT2) on PhIP-induced prostate cancer, we constructed a transgenic mouse model that targeted expression of human NAT2 to the prostate. Following construction, prostate, liver, lung, colon, small intestine, urinary bladder, and kidney cytosols were tested for human NAT1- and NAT2-specific N-acetyltransferase activities. Human NAT2-specific N-acetyltransferase activities were 15-fold higher in prostate of transgenic mice versus control mice, but were equivalent between transgenic mice and control mice in all other tissues tested. Human NAT1-specific N-acetyltransferase activities did not differ between transgenic and control mice in any tissue tested. Prostate cytosols from transgenic and control mice did not differ in their capacity to catalyze the N-acetylation of 2-aminofluorene, the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-PhIP or the N,O-acetylation of N-hydroxy-2-acetylaminofluorene. Transgenic and control mice administered PhIP did not differ in PhIP-DNA adduct levels in the prostate. This study is the first to report transgenic expression of human NAT2 in the mouse. The results do not support a critical role for bioactivation of heterocyclic amine carcinogens by human N-acetyltransferase-2 in the prostate. However, the lack of an effect may relate to the level of overexpression achieved and the presence of endogenous mouse acetyltransferases and/or sulfotransferases.

Animals↗

PubMed1999

A transgenic model of acetaldehyde overproduction accelerates alcohol cardiomyopathy.

Chronic alcohol consumption produces alcoholic heart muscle disease (AHMD), a prevalent form of congestive heart failure. Several hypotheses have been proposed to explain the damaging effects of alcohol on the heart, but neither the mechanism nor the ultimate toxin has been established. In this study, we use transgenic overexpression of alcohol dehydrogenase to elevate cardiac exposure to acetaldehyde, the major and most reactive metabolite of alcohol. Overexpression of alcohol dehydrogenase by 40-fold produced no detectable deleterious effects to the heart in the absence of alcohol. In the presence of alcohol, transgenic hearts contained 4-fold higher acetaldehyde than control hearts. Chronic alcohol exposure produced many changes similar to AHMD in transgenic hearts. Compared with control hearts, these pathological changes occurred more rapidly and to a greater extent: alcohol-exposed transgenic hearts were almost twice as large as control hearts. They demonstrated ultrastructural damage consistent with AHMD and had much lower contractility than alcohol-exposed control hearts. In addition, the transgenic hearts showed greater changes in mRNA expression for alpha-skeletal actin and atrial natriuretic factor than alcohol-exposed control hearts. Alterations in NAD(+)/NADH levels were insufficient to account for such severe damage in cardiomyopathic hearts. The increased damage produced in transgenic hearts suggests an important role for acetaldehyde in AHMD.

Acetaldehyde↗

PubMed1998

Catalase transfection decreases hydrogen peroxide toxicity in a pancreatic beta cell line.

BetaTC6-F7 cells like normal Beta cells were found to be highly sensitive to hydrogen peroxide and to possess very low levels of catalase. Therefore we tested whether overexpression of catalase could enhance resistance to hydrogen peroxide. Enzyme activity was increased forty fold by transient transfection of a catalase transgene. To assess protection from hydrogen peroxide a cotransfection method using a human growth hormone reporter gene was developed. Human growth hormone secretion was shown to be a suitable marker for insulin secretion since both hormones demonstrated virtually identical glucose dose response curves. Catalase transfection was found to provide significant protection against hydrogen peroxide indicating that low catalase may contribute to the sensitivity of cells to hydrogen peroxide.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Ultrastructural and functional analyses of nephropathy in calmodulin-induced diabetic transgenic mice.

BACKGROUND: Previous animal models of diabetic nephropathy have used diabetic animals for which the underlying defect was either uncertain or the diabetes was induced by potentially specific toxins. In this report, we describe the renal abnormalities in a transgenic mouse model that develops early-onset diabetes due to overexpression of calmodulin in pancreatic beta cells. METHODS: Renal tissues were collected from normal and transgenic mice at 112, 182, and 300 days. These were prepared for light microscopic observation, stained with polyethylenimine (for anionic sites), or rendered acellular by detergent extraction prior to observation by transmission and scanning electron microscopy. Morphometric analysis of glomerular basement membrane thickness was carried out by the "orthogonal intercept" method. Twelve-hour urine samples of fed and fasting mice were collected for urine volume and glucose and protein analyses. Blood glucose, blood urea nitrogen, serum insulin, and creatinine were determined in 60-90-day-old and 255-day-old mice by established methods. RESULTS: Morphometric analyses revealed age-related and transgene-related increases in glomerular basement membrane thickness. A 22% increase in transgenic diabetics over controls was seen at 112 days of age that developed to increases of 43% and 37% at 182 and 300 days of age, respectively. Mesangial matrix area was also increased markedly in transgenic mice. Surprisingly, even in the oldest diabetic mice, there was no reduction in anionic sites. Moreover, despite an eightfold increase in urine volume, these mice did not become significantly proteinuric. CONCLUSIONS: These results indicate that proteinuria of diabetes may be delayed or prevented by maintenance of a normal complement of glomerular basement membrane anionic sites. They also demonstrate that transgenic mice can provide a valuable model for discriminating between different aspects of diabetic nephropathy.

Animals↗

PubMed1996

Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice.

Weak antioxidant capacity, particularly low catalase activity in the heart, may be a factor responsible for the high sensitivity of this organ to doxorubicin-induced oxidative damage. To test this hypothesis, a heart-specific promoter was used to drive the expression of murine catalase cDNA in transgenic mice. Fifteen healthy transgenic mouse lines were produced. Cardiac catalase activity was constitutively overexpressed in both atrium and ventricule, ranging from 2- to 630-fold higher than normal. This enzyme activity was not altered in liver, kidneys, lungs, and skeletal muscles. Other antioxidant components, including glutathione, glutathione peroxidase, glutathione reductase, metallothionein, and superoxide dismutase, were not altered in the catalase-overexpressing heart. Mice (7 weeks old) from several transgenic lines and from nontransgenic controls were treated intraperitoneally with doxorubicin at a single dose of 20 mg/kg and sacrificed on the 4th day after treatment. As compared to normal controls, transgenic lines expressing catalase activity 60- or 100-fold higher than normal exhibited a significant resistance to doxorubicin-induced cardiac lipid peroxidation, elevation of serum creatine phosphokinase, and functional changes in the isolated atrium. Interestingly, 200-fold or greater elevation of catalase activity did not provide protection. The results provide direct evidence for the role of catalase in doxorubicin cardiotoxic responses.

Animals↗

PubMed1996

Unequal potency of transgenic yeast hexokinase on pancreatic beta cell metabolism and secretion.

Recent reports have proposed that sequence specific interactions between glucokinase and other beta cell proteins are important to glucokinase regulation of beta cell activity. We have previously reported enhancement of beta cell function by a transgenic hexokinase derived from yeast which has only 30% amino acid sequence homology to glucokinase. To test the functional significance of the amino acid sequence of islet glucokinase we have made a quantitative study of the effect of yeast hexokinase on beta cell glucose metabolism and insulin secretion. Transgenic and normal islets were assayed for hexokinase activity, glucose usage and insulin secretion. Most parameters were measured at six glucose concentrations between 0.5 and 20 mM glucose. Transgenic islet hexokinase activity measured in islet extracts exceeded normal islet hexokinase activity by 31 to 77 percent at different glucose concentrations. At all glucose concentrations tested the percentage increase in transgenic glucose metabolism greatly exceeded the percentage increase in transgenic hexokinase activity. The increase in transgenic glucose metabolism produced a proportional reduction in the threshold for glucose stimulated insulin secretion. However, yeast hexokinase had little if any effect on the first phase of insulin secretion. The finding that metabolism was very sensitive to yeast hexokinase but first phase secretion was not, supports recent proposals that hexokinase and glucokinase may be physically and functionally separated in the beta cell.

Amino Acid Sequence↗

PubMed1996

Thermoregulatory and heat-shock protein response deficits in cold-exposed diabetic mice.

Cold-induced expression of heat-shock proteins (HSPs) has been suggested to facilitate thermogenesis in brown adipose tissue (BAT). However, the regulation of this response and the mechanism supporting this facilitation have not been established. Because of the significant role of insulin in maintaining BAT thermogenesis, we employed a transgenic mouse model of diabetes to investigate the regulation and function of HSPs in BAT thermogenesis. These transgenic mice overexpress a calmodulin minigene regulated by the rat insulin II promotor, resulting in severe diabetes characterized by elevated blood glucose and glucagon that coincides with reduced serum and pancreatic insulin. Body temperature (Tb) of diabetic mice dropped significantly faster during a 3-h cold exposure (6 degrees C) than Tb of similarly treated control littermates. Cold exposure resulted in increased levels of constitutive and inducible HSP70 transcripts in control mice, but only constitutive HSP70 mRNA transcripts were induced in diabetic mice. Diabetes did not affect uncoupling protein induction, but cold-induced expression of members of other HSP families was reduced. Correspondingly, heat-shock regulatory factors were not activated in diabetic mice even though these factors were present. Phenylephrine induced HSP70 expression in control and diabetic animals, indicating that alpha-receptor-coupled HSP induction remained intact in BAT of diabetic mice. Insulin replacement restored the Tb response of diabetic mice as well as the HSP response. From these results it is clear that physiological signals that regulate cold-induced activation of BAT also regulate HSP expression in this tissue. This diabetic model provides a novel system in which the HSP response to cold has been selectively knocked out, making it a useful tool for the study of HSP regulation and function in BAT.

Adipose Tissue, Brown↗

PubMed1996

[Structure and ultrastructure of the endocrine pancreas in diabetic transgenic mice].

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.

Animals↗

PubMed1995

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.

Animals↗