Metabolic patterns and insulin responsiveness of enlarging fat cells

The rate and pattern of glucose metabolism, basal lipolysis, and intracellular concentration of free fatty acids were determined in isolated epididymal fat cell preparations (mean volume 30-800 pl) from rats on the basis of fat cell number and in relation to the cell volume. The effects of increasing glucose concentrations in the medium and of insulin on the cellular metabolic activities were compared. Expanding fat cell volume correlated positively and significantly (P < 0.001) with the synthesis of glyceride glycerol from glucose (correlation coefficient, r = 0.919), with rates of basal lipolysis (r = 0.663), and with intracellular free fatty acid accumulation (r = 0.796); it correlated negatively and significantly with glucose conversion to glyceride fatty acids (r = -0.814, P < 0.01). The differences in patterns of glucose metabolism and basal lipolysis between small (<100 pl) and large (>400 pl) fat cells were not modified by insulin or by increments in glucose concentration. The results indicate that the reduced capacity of the large fat cells to respond to insulin cannot be attributed solely to a limited capacity of the cells to take up and metabolize increasing amounts of glucose. The acquired unresponsiveness of the large cells to insulin may result from an alteration in the mechanism of action of insulin and may be related to an intracellular metabolic derangement with increased basal lipolysis, free fatty acid accumulation, and accelerated glyceride synthesis resulting from the accumulation of triglyceride.     

Adenosine and oxytocin reverse antagonism of cyclic AMP elevating agents to insulin activation of adipocyte pyruvate dehydrogenase

ACTH, isoprenaline, forskolin, and dibutyryl cyclic AMP prevented insulin from stimulating adipocyte pyruvate dehydrogenase in the presence of adenosine deaminase. Antagonism was reversed by N6-phenylisopropyladenosine as well as oxytocin. The stimulatory effects of insulin, adenosine and oxytocin on adipocyte pyruvate dehydrogenase appear to be through (a) mechanism(s) which is (are) similar or related.     

TPA inhibits insulin stimulated PIP hydrolysis in fat cell membranes: evidence for modulation of insulin dependent phospholipase C by proteinkinase C

Proteinkinase-C (PKC) stimulating phorbolesters induce in vitro insulin resistance of isolated adipocytes. This effect might be explained by an inhibition of insulin signal transduction at the level of the insulin receptor kinase. There is now some evidence that a phospholipase C is a potential candidate as a signal transducer at the postreceptor level. In order to determine whether phorbol esters might inhibit insulin signalling also at the level of a phospholipase C, we studied the insulin dependent [3H] phosphatidylinositol 4-monophosphate (PIP) hydrolysis of fat cell membranes. PIP hydrolysis was measured after in vitro stimulation with and without insulin. Insulin stimulated PIP hydrolysis in a dose dependent way. When plasma membranes from phorbolester (TPA) treated fat cells were used, this insulin stimulated phospholipase C activity was suppressed, provided, membranes have been prepared in a buffer containing serine phosphatase inhibitors. These data suggest that fat cell membranes contain an insulin dependent phospholipase C which is inhibited by TPA most likely via serine phosphorylation through proteinkinase C.     

White fat cell alpha-adrenergic receptors and responsiveness in altered thyroid status

[³H]-Dihydroergocryptine was used to identify α-adrenergic receptors in a crude adipocyte membrane fraction obtained from hyperthyroid, hypothyroid and euthyroid hamsters. Hyperthyroidism produced a 35–45 % decrease in the number of both the high and the low affinity [³H]-dihydroergocryptine binding sites but failed to affect the respective affinities of both these sites. On the other hand, binding of [³H]-dihydroergocryptine was unaltered in membranes of hypothyroid animals. Hamster adipocyte α-adrenergic responsiveness, reflected by the increment of epinephrine-stimulated cyclic AMP synthesis induced by phentolamine, was 50 % reduced by hyperthyroidism but unchanged by hypothyroidism. These results which demonstrate that thyroid hormones can regulate the density of adipocyte α-adrenergic receptors, suggest that in human fat cells where catecholamines produce opposite α- and β-adrenergic effects on lipolysis, the increased α-adrenergic responsiveness found in hyperthyroidism could be due, at least in part, to a reduction in the number of α-adrenergic receptors.     

Adenosine release from isolated rat adipocytes: influence of fat cell concentration and cell size

The release of adenosine by isolated rat adipocytes into the incubation medium was studied in relation to fat cell size and concentration. Incubations were carried out for 60 min at 37 degrees C in Krebs-Ringer bicarbonate-albumin medium containing 6 mM glucose. 2'-Deoxycoformycin was added to inhibit endogenous adenosine deaminase activity (maximal suppression was achieved at 0.8 microM concentration of the inhibitor). The data show that (a) the amount of adenosine released into the medium was similar for the first and second 30-min incubation periods; (b) increasing adipocyte concentration markedly inhibited adenosine release; and (c) large fat cells (volume greater than 300 pl) released significantly more adenosine (per fat cell) into the medium than smaller fat cells (volume less than 180 pl) when incubated at concentrations of less than or equal to 350,000 cells/ml. Above this cell concentration, differences between adenosine release and cell size were not noted. Adenosine release by isolated rat adipocytes appears to be a precisely regulated process which is exquisitly sensitive to the number of fat cells in the incubation medium and, to a certain extent, to the adipocyte size.     

Coincident increases in oxytocin receptor expression and EMG responsiveness to oxytocin in the ovine cervix at oestrus

This study has localised oxytocin receptor (OTR) mRNA expression within the cervix of non-pregnant ewes and related this to changes in the sensitivity of the cervical musculature to administered oxytocin (OT) during the oestrous cycle by recording electromyographic (EMG) activity. Cervices were collected from 34 ewes at specified time points throughout the cycle. OTR mRNA expression was localised by in situ hybridisation and results were expressed as optical density measurements from autoradiographs in each of four different cervical regions. EMG recordings were made for up to 8 h per day from four non-pregnant ewes undergoing seasonal oestrous cycles between Days −3 and +3 relative to oestrus (Day 0). The highest concentrations of OTR mRNA were detectable within the luminal epithelium (LE) of the cervix, with values increasing from Day 15 of the cycle, peaking during the follicular phase (P<0.001, compared to the mid-luteal phase) and returning to basal by Day 2. There was a small but significant increase in OTR mRNA hybridisation (above basal/luteal phase values) within the stromal cells (STR) adjacent to the lumen (P<0.05) during the same time period, but no differences from basal values were detectable in the dense collagenous annular ring or in tissue superficial to this. Analysis of pooled EMG activity recorded daily from the cervix indicated that endogenous contractile activity was higher on Day 0 than on the Days +1 (P<0.05), −2, +2 and +3 (P<0.001). The response to bolus intravenous (i.v.) injections of 25 mU OT (25 mU) varied with day of the cycle. This dose produced a measurable and significant response on Days 0 (P<0.001) and +1 (P<0.001), but not on any of the other days, indicating that the sensitivity of the cervical musculature to OT peaked on these days. These data show that the cervix is highly responsive to OT at oestrus. This coincides with an increase in OTR mRNA expression in the luminal epithelial cells, suggesting the likely production of an intermediary messenger between the epithelial and smooth muscle cells.     

Nicotinamide modulation of rat pancreatic islet cell responsiveness in vitro.

The influence of nicotinamide (NA), a highly suitable precursor substrate for NAD synthesis in various tissues, on islet cell responsiveness was determined. After a 30 minute perifusion with this compound, nicotinamide, in a dose-dependent manner, potentiated glucose-induced insulin secretion. Maximal potentiation (approximately 250%) was observed at 20 mM NA and the threshold for potentiation was 3 mM. In the absence of glucose, NA did not affect basal secretion rates. Mannoheptulose blocked the primary stimulant action of glucose and the potentiating effects of NA. NA did not alter the rate of glucose usage by isolated islets. These results further underscore the possible importance of pyridine nucleotides in stimulated secretion.     

Differential responsiveness to oxytocin of the uterus and cervix in the ovariectomized ewe

The responsiveness of the uterus and cervix to oxytocin was compared in ovariectomized ewes fitted with intraparietal electrodes and treated with 17β-oestradiol. Before the injection of the steroid, only the cervix presented dose-related responses to oxytocin infusions. Within the 3–5-day period after oestrogen injection, both the uterus and cervix presented almost similar responsiveness to the neurohormonal agent. After the 6th day following oestrogen, the cervix remained reactive to the infusions of oxytocin, whereas the uterus failed to respond. It is suggested that the reactivity of the cervix, which is at least partially independent of oestrogen priming, may be due to oestrogen-independent oxytocin receptors present only in the cervix.     

A theoretical approach to G-protein modulation of cellular responsiveness

 Structure and function of cells often depend critically on molecular signals arriving at their surface. There are universal mechanisms of signal transduction and signal processing across cell membranes. In this paper the mechanisms involving guanine-nucleotide regulatory proteins (“G-proteins”) and certain receptor-kinases are considered. On the basis of recent findings in molecular biology a mathematical model is developed taking into account all essential components in the biochemical network between first and second messenger. There are two coupled feedback loops inherent in this process. The model finally consists of three nonlinear equations, which are obtained from a system of originally ten equations by using conservation laws and quasi-steady state conditions. The second part of the paper contains a mathematical analysis of the model. Solutions describing the temporal development of the involved biochemical species are shown to be bounded, more specifically to remain, independent of the size of the input signal, in a bounded domain of the state space. For the situation of stationary input signals existence, uniqueness and asymptotic stability of steady states are derived. We also demonstrate biologically relevant stimulus-response properties like monotonicity and saturation effects. For temporally non-constant input signals we show numerically that the model is able to produce phenomena of hypersensitivity and desensitization which are important characteristics of cellular responsiveness. Received 18 March 1996; received in revised form 15 April 1996     

Ovine trophoblast protein-one inhibits development of endometrial responsiveness to oxytocin in ewes

In experiment (Exp) 1, 12 cyclic ewes had catheters placed into each uterine horn on Day 7 (estrus = Day 0). On Days 11-15, 6 ewes received twice-daily intrauterine infusions of 1.5 mg serum protein (SP) into each uterine horn and 6 ewes received infusions of 1.08 mg SP + 0.42 mg ovine conceptus secretory proteins (oCSP) containing 25 micrograms ovine trophoblast protein-one (oTP-1) as determined by radioimmunoassay (25-35% bioactive by antiviral assay). SP-infused and oCSP-infused ewes had similar plasma 13,14-dihydro-15-keto prostaglandin F2 alpha (PGF2 alpha) profiles in response to oxytocin on Day 11, but SP ewes became more responsive (p less than 0.01) to oxytocin on Days 13 and 15 than oCSP ewes. SP ewes also had greater incorporation of [3H]inositol into inositol trisphosphate (IP3) (+3449%, p less than 0.01) and total inositol phosphate (IP) (+760%, p less than 0.08), in response to oxytocin, than did oCSP ewes (+553 and +168% for IP3 and total IP, respectively) in endometrium collected at ovariectomy/hysterectomy on Day 16. Mean CL weights on Day 16 and mean concentrations of progesterone in plasma collected at 12-h intervals on Days 6-16 were not different for SP and oCSP ewes, but concentrations of progesterone were lower (p less than 0.05) in SP ewes on Days 15-16 than for oCSP ewes. These results indicate that oTP-1 may prevent luteolysis by inhibiting development of endometrial responsiveness to oxytocin and, therefore, reduce oxytocin-induced synthesis of IP3 and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Congruence of individual responsiveness to dietary cholesterol and to saturated fat in humans

Previous experiments have shown that differences between humans in the response of serum cholesterol to dietary cholesterol are at least partly reproducible and stable over a prolonged period. In this study it was investigated whether enhanced sensitivity to dietary cholesterol and saturated fat go together. The subjects had also participated in three or four experiments dealing with the reproducibility of the effect on blood cholesterol of either adding cholesterol to the diet in normal subjects (NORM-EGG group; n = 23) or of cessation of egg consumption in subjects with a high habitual egg intake (HAB-EGG group; n = 24). In the present experiment the NORM-EGG subjects were fed a mixed natural diet providing 21% of energy as polyunsaturated and 11% as saturated fat (P/S2 ratio, 1.9) for 3 weeks, and one providing 5% of energy as polyunsaturated fat and 23% as saturated fat (P/S ratio, 0.2) for the next 3 weeks. The HAB-EGG group was fed the same diets in reverse order. The serum cholesterol concentrations were higher on the low P/S diet than on the high P/S diet (on average 23% in normal subjects and 16% in habitual egg eaters). The correlation coefficient between each subject's serum cholesterol response to fatty acids and his or her average response to dietary cholesterol in the dietary cholesterol experiments was 0.62 for the normal subjects (P less than 0.01) and 0.15 for the HAB-EGG group. We conclude that modest differences in responsiveness of serum cholesterol to dietary saturated fat do exist in humans, and that, in people of normal cholesterol intake, responsiveness to dietary cholesterol and to saturated fat tend to go together.     

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.     

Alterations in the responsiveness of diabetic fibroblasts to insulin

Fibroblastic cultures from the skin of nondiabetic and diabetic (db/db) mice have been used to investigate alterations in the biological responses of diabetic cells to insulin. Confluent cultures from the skin of both nondiabetic and diabetic animals possess specific receptors for insulin. Diabetic fibroblasts exhibit only 36% as much specific binding of insulin as nondiabetic fibroblasts, because of a decrease in the total number of binding sites, without a change in binding affinity. Insulin caused a time- and dose-dependent increase in the rate of 2-deoxy D-glucose (dGlc) uptake and in ornithine decarboxylase (ODC) activity of both nondiabetic and diabetic fibroblasts. In nondiabetic cells, half-maximal increase in dGlc uptake was obtained with 0.3 nM insulin, and a maximum increase of 120% was obtained with 4.1 nM insulin. In contrast, diabetic cultures required 0.8 nM insulin for a half-maximal increase in dGlc uptake, and maximum stimulation with 4.1 nM insulin was only 50% above control levels. With 4-fold higher insulin concentrations, ODC activity of diabetic cells was only 40% that of nondiabetic cells. In nondiabetic cells, down regulation of insulin receptors by insulin abolished the ability of insulin to stimulate dGlc uptake. These results demonstrate that cells cultured from diabetic animals, which possess a decreased number of insulin receptors, also exhibit decreased stimulation of deoxy D-glucose uptake and ornithin decarboxylase activity by insulin.