Prevention of insulin resistance by environmental manipulation as young rats mature

This study was initiated in an attempt to see if the insulin resistance associated with maturation in young rats could be prevented by environmental manipulation. Consequently, seven week-old rats were either housed in standard laboratory cages and fed a calorie-restricted diet or placed individually in exercise wheel cages and allowed to eat chow ad lib. A control group of rats was housed in standard laboratory cages from seven weeks to five months of age, and also allowed to eat chow ad lib. When studied at five months of age, the chow-fed rats weighed more (624 +/- 8 g) than either the calorie restricted (479 +/- 9 g) or exercise trained (485 +/- 13 g) rats. Insulin action was compared in the three groups by assessing the steady-state serum glucose (SSSG) and insulin (SSSI) concentrations achieved during a continuous intravenous infusion of glucose and exogenous insulin. The results of these studies indicated that SSSG concentration was significantly higher (P less than 0.001) in chow-fed rats than in the two experimental groups. Since SSSI concentrations were the same in all three groups, lower SSSG concentrations in calorie-restricted and exercise trained rats indicates that insulin-stimulated glucose uptake was preserved in these two groups as compared to the chow-fed population. In an attempt to understand why exercise training and calorie restriction prevented the development of insulin resistance, muscle glycogen synthase activity and muscle capillary density were compared in the three groups of five month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a GA transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts from a compound heterozygote for this transition (and for a deletion mutation) produce no detectable HEXA mRNA. The intron 7+1 mutation occurs in the base adjacent to the site of the adult-onset TSD mutation (G805A). In both mutations a restriction site for the endonuclease EcoRII is abolished. Unambiguous diagnosis, therefore, requires allele-specific oligonucleotide hybridization to distinguish between these two mutant alleles. The intron 7+1 mutation has been detected in three unrelated families. Obligate heterozygotes for the intron 7+1 mutation were born in the Saguenay-Lac-St-Jean region of Quebec. The most recent ancestors common to obligate carriers of this mutation were from the Charlevoix region of the province of Quebec. This mutation thus has a different geographic centre of diffusion and is probably less common than the exon 1 deletion TSD mutation in French Canadians. Neither mutation has been detected in France, the ancestral homeland of French Canada.     

Evidence for age-related changes in pyridine nucleotide content of isolated rat islets

The purpose of this study was to document the effect of age on alpha-glycerophosphate activity and pyridine nucleotide concentration in pancreatic islets isolated from rats. In order to do this, islets were isolated from pancreases of 2 and 12 month-old rats, and measurements made of alpha-glycerophosphate activity and of NAD+ and NADH, determinations were made following incubation at both basal (5.6 mM) and elevated glucose concentrations (28 mM). The results indicated that islet alpha-glycerophosphate dehydrogenase activity was decreased (P less than 0.001) by approximately 50% in the older rats. This was associated with an increase in mean (+/- SEM) basal NADH content (pmol/microgram DNA) in 12 month-old (4.48 +/- 0.31) as compared to 2 month-old rats (2.73 +/- 0.49). Although mean (+/- SEM) basal NAD+ levels (pmol/microgram DNA) were the same in 2 and 12 month-old rats (29.4 +/- 2.5 and 30.8 +/- 2.8, respectively), NAD+ content following incubation at elevated levels of glucose declined (absolutely and relatively) to a significantly greater degree in the younger rats. The incremental rise in islet NADH concentration following incubation at the elevated glucose concentration was similar in the two groups, but the relative increase was only approximately half as great in islets from 12 month-old rats. These data indicate that the age-related decline in the activity of alpha-glycerophosphate dehydrogenase, the enzyme regulating the glycerophosphate shuttle system in 12 month-old rats, is associated with alterations in islet pyridine nucleotide composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of isoenzymic forms of hepatic Ca2+-activated-phospholipid-dependent protein kinase in various animal models

We have examined the protein kinase C that are present in mouse, rat, guinea pig and rabbit liver. Initial subcellular fractionation analysis indicated that the majority (75-85%) of the activity was associated with particulate fraction of the liver. The bound protein kinase C was dissociated by homogenization of livers in buffer containing EGTA, EDTA and various proteolytic inhibitors and the solubilized extract was used to resolve multiple forms of the enzyme. The fractionation procedure, sequentially utilized (NH4)2SO4 precipitation, ion exchange chromatography, gel permeation chromatography, and hydroxylapatite column chromatography. With hydroxylapatite, protein kinase C was resolved into three isoenzymic forms designated C-I, C-II and C-III. In each case, the predominant activity consisted of C-II and C-III and together they represented about 80-88% of the total activity. All three isoenzymes from each source demonstrated an absolute requirement for PS + Ca2+ (approximately 25-50 fold stimulation over basal activity); for maximal activity the isoenzymes also required the presence of divalent metal ion, Mg2+ (5-10 mM) and lysine rich histone (H1). Both diolein and TPA decreased the Ca2+ and PS requirement of the enzyme and directly stimulated enzyme activity in the presence of suboptimal concentrations of Ca2+ and PS. In conclusion, the present studies suggest that protein kinase C in mammalian liver exists in isoenzymic forms.     

Effect of adrenalectomy on in vivo glucose metabolism in insulin resistant Zucker obese rats

Lean (Fa/?) and obese (fa/fa) Zucker rats were adrenalectomized (ADX) in order to assess the contribution of adrenal hormones to insulin resistance of the obese Zucker rat. Glucose utilization was measured using an insulin suppression test. Sham-operated obese rats gained almost twice as much weight as sham-operated lean littermates. However, body weight gain of ADX animals was comparable in both genotypes. It was significantly less than that of the respective sham-operated controls. Body weight differences can be accounted for almost entirely by a marked loss of adipose tissue. Although insulin resistance may be attributable to obesity in part, steroid hormones are thought to be directly antagonistic to insulin for glucose metabolism. Adrenalectomy resulted in a decrease in serum glucose concentrations for both lean and obese Zucker rats compared with their respective sham-operated groups. Serum insulin concentration of lean ADX rats was 23% of sham-operated controls; in obese ADX rats, it was 9% of controls. Elevated levels of steady state serum glucose (SSSG) levels in sham-operated obese rats demonstrate a marked resistance to insulin induced glucose uptake compared with sham-operated lean animals. Adrenalectomy caused a marked improvement in insulin sensitivity of obese rats. The hyperglycemic SSSG levels of the obese rats were reduced 2.5 times by ADX. These results indicate that insulin resistance of Zucker obese rats can be ameliorated by ADX, suggesting adrenal hormones contribute to insulin resistance in these animals.     

Is the insulin resistance of patients with noninsulin-dependent diabetes mellitus secondary to insulin deficiency?

Defects in both insulin secretion and action have been documented in patients with noninsulin-dependent diabetes mellitus (NIDDM), leading to the suggestion that both fasting hyperglycemia and insulin resistance in NIDDM are secondary to insulin deficiency. In order to test this hypothesis, insulin secretion (plasma insulin response to oral glucose) and insulin action (insulin clamp) were determined in 25 patients with NIDDM. The results documented relationships between incremental plasma insulin response to glucose and degree of fasting hyperglycemia (r = -.045, P less than 0.05) and insulin-stimulated glucose utilization (r = 0.25, P = NS). These data indicate that differences in insulin secretory response accounted for only approximately 20% of the variance in fasting plasma glucose level and 6% of the variance in insulin resistance in NIDDM. Thus, differences in insulin-secretory response contribute modestly to magnitude of glycemia, and not at all to variations in insulin resistance in NIDDM, permitting rejection of the hypothesis that insulin resistance is secondary to insulin deficiency.     

Regulation of luteal cell 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by estradiol

Recent studies have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular sterol availability and metabolism. This investigation was performed to examine the effect of estradiol on de novo synthesis of cholesterol. Pregnant rats hypophysectomized and hysterectomized on Day 12 were treated for 72 h with either estradiol or testosterone. De novo cholesterol synthesis was determined by measurement of the specific activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis, in microsome-enriched preparations of luteal tissue and incorporation of [14C] acetate into cholesterol by corpora lutea incubated in vitro. Estradiol or testosterone treatment caused a 4- to 5-fold stimulation of luteal cholesterol biosynthesis, as measured by these techniques. NaF, an inhibitor of phosphatase which blocks the conversion of the inactive enzyme to the active form, reduced the HMG CoA reductase activity to 30% in corpora lutea obtained from either steroid or vehicle-treated rats. However, an increase in enzyme activity of comparable magnitude by steroids was observed whether microsomes were isolated with or without NaF. The effect of estradiol appears to be enzyme-specific, since it failed to affect the microsomal marker, NADPH-cytochrome c reductase. Since the cholesteryl ester content of corpora lutea falls in response to steroid treatment, rats were treated with 4-aminopyrazolo-[3,4d]pyrimidine (4-APP) to deplete cellular cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)     

The innervation of the pyloric region of the crab,Cancer borealis: Homologous muscles in decapod species are differently innervated

Summary The muscles of the pyloric region of the stomach of the crab,Cancer borealis, are innervated by motorneurons found in the stomatogastric ganglion (STG). Electrophysiological recording and stimulating techniques were used to study the detailed pattern of innervation of the pyloric region muscles. Although there are two Pyloric Dilator (PD) motorneurons in lobsters, previous work reported four PD motorneurons in the crab STG (Dando et al. 1974; Hermann 1979a, b). We now find that only two of the crab PD neurons innervate muscles homologous to those innervated by the PD neurons in the lobster,Panulirus interrruptus. The remaining two PD neurons innervate muscles that are innervated by pyloric (PY) neurons inP. interruptus. The innervation patterns of the Lateral Pyloric (LP), Ventricular Dilator (VD), Inferior Cardiac (IC), and PY neurons were also determined and compared with those previously reported in lobsters. Responses of the muscles of the pyloric region to the neurotransmitters, acetylcholine (ACh) and glutamate, were determined by application of exogenous cholinergic agonists and glutamate. The effect of the cholinergic antagonist, curare, on the amplitude of the excitatory junctional potentials (EJPs) evoked by stimulation of the pyloric motor nerves was measured. These experiments suggest that the differences in innervation pattern of the pyloric muscles seen in crab and lobsters are also associated with a change in the neurotransmitter active on these muscles. Possible implications of these findings for phylogenetic relations of decapod crustaceans and for the evolution of neural circuits are discussed.Abbreviations ACh acetylcholine - Carb carbamylcholine - cpv muscles of the cardio-pyloric valve - cpv7n nerve innervating muscle cpv7 - cv muscles of the ventral cardiac ossicles - cv1n nerve innervating muscle cvl - cv2n nerve innervating muscle cv2 - EJP excitatory junctional potential - IC inferior cardiac neuron - IV inferior ventricular neuron - IVN inferior ventricular nerve - LP lateral pyloric neuron - LPG lateral posterior gastric neuron - lvn lateral ventricular nerve - mvn medial ventricular nerve - p muscles of the pylorus - PD pyloric dilator neuron - PD _in intrinsic PD neuron - PD _ex extrinsic PD neuron - pdn pyloric dilator nerve - PY pyloric neuron - pyn pyloric nerve - STG stomatogastric ganglion - VD ventricular dilator neuron     

Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells

Electron microscopic studies of perfused rat adrenals indicate that plasma lipoproteins become concentrated in a specialized cell surface compartment called microvillar channels. Closely associated plasma membranes of sinusoidal microvilli of zona fasciculata cells form channels that normally are filled with electron dense particles the size of high density lipoproteins (HDL). In rats made acutely deficient in plasma lipoproteins (by treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) for 1 day), particles within the microvillar channels are decreased in number. When adrenal glands of these rats are perfused with media lacking plasma lipoproteins, many but not all of these HDL-like particles are washed out. However, when these adrenals are perfused with large amounts (100-500 micrograms protein/ml) of HDL, microvillar channels become packed with electron dense particles similar to those found in vivo. These microvillar channels become wider and filled with larger particles when low density lipoproteins (LDL) are perfused through the adrenals. Autoradiograms of 125I-labeled HDL-perfused adrenals show silver grains specifically associated with the cell surface microvillar channels, and confirm the notion that the particles filling the channels are exogenously delivered HDL. Physiologic data from similarly perfused adrenals in a parallel study show that the channel-refilling process is directly related to selective (i.e., nonendocytic) cholesterol uptake and that this cholesterol uptake is associated with corticosterone production. Together, these data suggest the hypothesis that plasma lipoprotein cholesterol utilized for corticosteroid synthesis in rat adrenal fasciculata cells may be derived from lipoproteins trapped in surface-associated microvillar channels. Although the mechanism responsible for the cholesterol transfer is not yet defined, it is clearly distinct from the classical process of receptor-mediated endocytosis and catabolism of lipoprotein particles.