Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene.

The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly. Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila.

Glucose transporter GLUT3: ontogeny, targeting, and role in the mouse blastocyst.

The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump fluid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.

Differential targeting of facilitative glucose transporters in polarized epithelial cells.

We have examined the intracellular localization of five facilitative glucose transporter proteins, one endogenous (GLUT-1) and four exogenous (GLUT-2, -3, -4, and -5), in polarized epithelial cells. GLUT-2, -3, -4, and -5 were stably transfected into Madin-Darby canine kidney (MDCK) cells, and peptide-specific antibodies were used to establish their distribution by immunofluorescence and immunoelectron-microscopic techniques. GLUT-1 and -2 were predominantly targeted to the basolateral domain of the cell, whereas GLUT-3 and -5 were targeted to the apical plasma membrane. The insulin-regulatable glucose transporter GLUT-4 was found in intracellular tubulovesicular structures beneath the surface of the cell. Vectorial 2-deoxy-D-glucose uptake measurements revealed that approximately 95% of glucose entry into wild-type MDCK cells occurs via the basolateral membranes. In GLUT-3-transfected cells, however, apical glucose uptake increased to approximately 55%; this was not observed in cells expressing the other GLUT isoforms. The discrete and differential intracellular localizations of the various GLUTs, in addition to the high level of sequence homology and predicted secondary structure similarity, render the GLUT family ideal for the study of intrinsic targeting motifs involved in the establishment and maintenance of cellular polarity.

Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.

A method for the production of embryonic stem (ES) cell-embryo chimeras was developed that involves the simple coculture of eight-cell embryos on a lawn of ES cells. After coculture, the embryos with ES cells attached are transferred to normal embryo culture medium and allowed to develop to the blastocyst stage before reimplantation into foster mothers. Although the ES cells initially attach to the outside of the embryos, they primarily colonize the inner cell mass and its derivatives. This method results in the efficient production of chimeras with high levels of chimerism including the germ line. As embryos are handled en masse and manipulative steps are minimal, this method should greatly reduce the time and effort required to produce chimeric mice.

Insulin action and determinants of glycaemia in a rat model of type 2 (non-insulin-dependent) diabetes mellitus.

We aimed to assess prandial responses, basal glucose turnover and insulin action (euglycaemic clamp) in a very low-dose neonatal streptozotocin model of Type 2 (non-insulin-dependent) diabetes mellitus. Male Wistar rats were injected at 2 days of age with 45 mg/kg streptozotocin or vehicle (control). At 8 weeks, the groups were subdivided and fed either a high-fat or high-starch diet for 3 weeks. Both the fat diet and streptozotocin treatments had independent hyperglycaemic effects (streptozotocin/fat 9.3 +/- 0.3 mmol/l; streptozotocin/starch 7.5 +/- 0.3 mmol/l; control/fat 7.4 +/- 0.1 mmol/l; all p less than 0.01 vs control/starch 6.4 +/- 0.1 mmol/l). The fat diet effect was associated with both a reduction in basal glucose clearance (p less than 0.001) and in basal hepatic glucose output (p less than 0.05). Streptozotocin increased basal hepatic glucose output. Significantly higher prandial glycaemia in the streptozotocin/starch group occurred despite similar insulin levels and appeared to be related to an impaired early insulin response. Whole-body and tissue-specific insulin sensitivity were significantly depressed in fat-fed animals compared to starch-fed animals, however there were no significant effects of streptozotocin treatment. We conclude that fasting hyperglycaemia associated with abnormalities in both glucose production and clearance can exist in the presence of a basal hepatic glucose output which is reduced compared to control animals. Furthermore, dietary-fat-induced insulin resistance is not exacerbated by the relative insulin deficiency and/or mild hyperglycaemia observed when dietary fat and neonatal streptozotocin-treatments are combined.

Enhanced responses to stress induced by fat-feeding in rats: relationship between hypothalamic noradrenaline and blood glucose.

High-fat-feeding in rats has been reported to enhance stress reactions, as assessed by elevation of blood glucose and corticosterone levels. This study was designed to investigate the relationship between changes in blood glucose and hypothalamic neuronal noradrenaline activity (HNNA), as indexed by the ratio of dihydroxyphenylethyleneglycol (DHPG) to noradrenaline (NA), following physiological stress in high-fat-fed rats. Two groups of adult male Wistar rats were fed isocaloric diets high in fat (59% of calories) or starch (70% of calories). After 3 weeks each of these groups was further subdivided into (a) control, (b) 2 min ambient temperature (20 degrees C) swim or (c) 2 min swim in ice-cold water. Animals were decapitated 20 min after commencing the swim; trunk blood and a sample of medial basal hypothalamus were obtained. Computerized gas chromatography/mass spectrometry was used to measure hypothalamic DHPG and NA concentrations. There were no differences between fat- and starch-fed rats in basal levels of serum glucose, insulin or corticosterone and no differences in DHPG, NA or DHPG/NA ratio. Compared to starch-fed rats, ambient swim stress in the fat-fed group produced significantly larger serum glucose (P less than 0.01), serum corticosterone (P less than 0.05), DHPG (P less than 0.05) and DHPG/NA (P less than 0.01) responses. Following cold swim stress similar differences between fat- and starch-fed animals were observed. In addition, serum insulin was found to be significantly suppressed in the fat-fed group (P less than 0.05) following cold swim.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid.

High levels of some but not all dietary fats lead to insulin resistance in rats. The aim of this study was to investigate the important determinants underlying this observation. Insulin action was assessed with the euglycemic clamp. Diets high in saturated, monounsaturated (omega-9), or polyunsaturated (omega-6) fatty acids led to severe insulin resistance; glucose infusion rates [GIR] to maintain euglycemia at approximately 1000 pM insulin were 6.2 +/- 0.9, 8.9 +/- 0.9, and 9.7 +/- 0.4 mg.kg-1. min-1, respectively, versus 16.1 +/- 1.0 mg.kg-1.min-1 in chow-fed controls. Substituting 11% of fatty acids in the polyunsaturated fat diet with long-chain omega-3 fatty acids from fish oils normalized insulin action (GIR 15.0 +/- 1.3 mg.kg-1.min-1). Similar replacement with short-chain omega-3 (alpha-linolenic acid, 18:3 omega 3) was ineffective in the polyunsaturated diet (GIR 9.9 +/- 0.5 mg.kg-1.min-1) but completely prevented the insulin resistance induced by a saturated-fat diet (GIR 16.0 +/- 1.5 mg.kg-1.min-1) and did so in both the liver and peripheral tissues. Insulin sensitivity in skeletal muscle was inversely correlated with mean muscle triglyceride accumulation (r = 0.95 and 0.86 for soleus and red quadriceps, respectively; both P less than 0.01). Furthermore, percentage of long-chain omega-3 fatty acid in phospholipid measured in red quadriceps correlated highly with insulin action in that muscle (r = 0.97). We conclude that 1) the particular fatty acids and the lipid environment in which they are presented in high-fat diets determine insulin sensitivity in rats; 2) impaired insulin action in skeletal muscle relates to triglyceride accumulation, suggesting intracellular glucose-fatty acid cycle involvement; and 3) long-chain omega-3 fatty acids in phospholipid of skeletal muscle may be important for efficient insulin action.

Inducement by fat feeding of basal hyperglycemia in rats with abnormal beta-cell function. Model for study of etiology and pathogenesis of NIDDM.

Non-insulin-dependent diabetes mellitus (NIDDM) is a heterogenous disorder characterized by defects in insulin action and secretion. This study was aimed at developing a rat model in which these pathogenic factors might be studied. Male Wistar rats were injected at 2 days of age with 45 or 30 mg/kg streptozocin (STZ) or vehicle (control). Fasting plasma glucose and insulin levels were not significantly different between the two groups between 5 and 8 wk of age. At 8 wk, half of each group was randomly assigned to isocaloric diets high in either fat (59% of calories) or starch (70% of calories). After 1 wk on the diets, 45-mg/kg-STZ-administered fat-fed animals displayed significant fasting hyperglycemia (8.6 +/- 0.2 mM; P less than 0.01), which was exacerbated by the stress of anesthesia and/or cannulation, whereas no changes were observed in any of the other groups before (STZ starch fed, 6.7 +/- 0.1 mM; control fat fed, 6.8 +/- 0.1 mM; control starch fed; 6.4 +/- 0.1 mM) or after anesthesia and/or cannulation. In the 30-mg/kg-STZ animals, fat feeding did not significantly elevate plasma glucose concentration, but a significant hyperglycemic response was seen with anesthesia and/or cannulation. In all STZ groups, substantial impairment of glucose-induced insulin secretion was observed, particularly early-phase insulin secretion. Further studies indicated that STZ animals on a diet conferring normal insulin sensitivity (starch) maintained basal normoglycemia and mildly impaired (i.v.) glucose tolerance despite this gross insulin secretory defect.(ABSTRACT TRUNCATED AT 250 WORDS)

2-deoxy-D-glucose-induced hyperglycemia: role for direct sympathetic nervous system activation of liver glucose output.

The hypothalamus plays an important integrative role in the control of peripheral metabolism, achieved by modulation of autonomic outflow to the endocrine pancreas, the liver and the adrenal medulla. This study examines the role of direct sympathetic nervous system control of hepatic glucose output during neuroglycopenia induced by the non-metabolizable glucose analogue 2-deoxy-D-glucose (2-DG). Steady-state tracer methodology was used to directly measure hepatic glucose output (Ra) in pentobarbitone-anesthetised male Wistar rats (220-320 g). Administration of 500 mg/kg 2-DG i.p. produced an increase in Ra from a control value of 7.3 +/- 0.3 mg/kg.min (n = 4) to 15.2 +/- 2.2 mg/kg.min-1 (n = 8), corresponding to an increase in plasma glucose (PG) from 6.4 +/- 0.1 mmol/l to 10.1 +/- 0.4 mmol/l. This rise was countered by the sympathetic noradrenergic blocker guanethidine (100 mg/kg i.p.), reducing Ra to 10.4 +/- 0.9 mg/kg.min-1 and PG to 6.1 +/- 0.3 mmol/l (n = 8), despite markedly lower plasma insulin (PI) levels (2-DG: PI = 94.7 +/- 18.6 mU/l (n = 7), 2-DG + guanethidine: PI = 41.4 +/- 3.3 mU/l (n = 8). Hyperglycemia and elevated liver glucose output were maintained in ADX animals treated with 2-DG, indicating an absence of adrenal-medullary influence (2-DG: Ra = 15.2 +/- 2.2 mg/kg.min-1, 2-DG + ADX = 15.6 +/- 1.0 mg/kg.min-1). Elevated Ra in the 2-DG + ADX was maintained despite markedly elevated insulin levels 349.3 +/- 72.6 mU/l (n = 7)).(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic noradrenergic and sympathoadrenal control of glycemia after stress.

Central noradrenergic pathways play a significant role in mediating blood glucose levels after neuroglycopenia. To further investigate hypothalamic noradrenergic neuronal activity (NNA) and sympathoadrenal influences in glucoregulation, we studied the effects of acute stress on glycemia and insulin release in normal and adrenalectomized (ADRX) rats. Within 5 min of exposure of rats to ether or cold-swim stress, significant positive correlations were evident between hypothalamic NNA and serum glucose levels (r = 0.70, P less than 0.001; at 15 min r = 0.78, P less than 0.0001). Five minutes after stress in the intact rat, insulin release was inhibited and serum insulin levels inversely correlated to hypothalamic NNA (r = 0.45, P less than 0.05). This relationship between insulin and NNA was no longer present 15 min after stress, but the levels of insulin remained inappropriately low with respect to the elevated serum glucose levels (approximately 30% above basal). Blockade of sympathetic noradrenergic pathways by treatment of intact rats with guanethidine prevented the rise in glucose after cold-swim stress but did not prevent the inhibition of insulin release. Fifteen minutes after exposure of ADRX rats to cold-swim stress their hypothalamic NNA and serum glucose levels were similar to intact animals. However, in contrast to their intact counterparts, serum insulin levels were significantly elevated (P less than 0.01). These data are consistent with central noradrenergic neural pathways directly mediating hepatic glucose release and indirectly inhibiting pancreatic insulin release via activation of adrenal medullary catecholamines.

Fish oil prevents insulin resistance induced by high-fat feeding in rats.

Non-insulin-dependent diabetes mellitus is an increasingly prevalent disease in Western and developing societies. A major metabolic abnormality of non-insulin-dependent diabetes is impaired insulin action (insulin resistance). Diets high in fat from vegetable and nonaquatic animal sources (rich in linoleic acid, an omega-6 fatty acid, and saturated fats) lead to insulin resistance. In rats fed high-fat diets, replacement of only 6 percent of the linoleic omega-6 fatty acids from safflower oil with long-chain polyunsaturated omega-3 fatty acids from fish oil prevented the development of insulin resistance. The effect was most pronounced in the liver and skeletal muscle, which have important roles in glucose supply and demand. The results may be important for therapy or prevention of non-insulin-dependent diabetes mellitus.