The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue- and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n=24) and lean (fa/-, n=24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (488+/-7 vs 315+/-9 g) with fasting hyperglycaemia (10.5+/-0.8 vs 7.7+/-0.1 mM) and hyperinsulinaemia (7167+/-363 vs 251+/-62 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (174+/-16 vs 108+/-12 pmol/min/mg protein, P<0.05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-theta and PKC-epsilon: e.g. PKC activity in the membrane fraction of muscle from obese animals was 283+/-42 (fed) vs 107+/-20 pmol/min/mg protein (fasting) compared with 197+/-27 (fed) and 154+/-21 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-theta and PKC-epsilon. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes.
X Qu, JP Seale, and R Donnelly
L. M. S. Carlsson, R. G. Clark, A. Skottner, and I. C. A. F. Robinson
The effects of streptozotocin-induced diabetes on weight gain, bone growth and GH secretion have been studied in conscious chronically cannulated male rats. In addition to the classic diabetic symptoms (hyperphagia, polydipsia, polyuria, glycosuria and hyperglycaemia), the slow body weight gain (0·95 ± 0·5 compared with 2·63 ± 0·5 g/day in non-diabetic controls) was associated with a reduction in bone growth (from 162 ± 9 to 48 ± 4 μm/day) and a reduced pituitary GH content (from 1·5 ± 0·2 to 0·6 ± 0·06 mg/gland). Serial blood sampling during the day or overnight showed that the normal male episodic GH secretory pattern was obliterated in the diabetic animals. The constant osmotic stimulation of hyperglycaemia and high fluid turnover was reflected in a significant reduction in pituitary oxytocin and arginine vasopressin (AVP) stores. Intravenous insulin infusions (67–1340 pmol/h for 4 or 7 days) caused a large initial weight gain (> 20 g in 2 days) followed by a slower increase, and stimulated tibial bone growth (to 100 ± 16 and 126 ± 8 μm/day after 4 or 7 days respectively). Insulin infusion for 7 days also increased pituitary GH content (to 1 ± 0·15 mg/gland), and the normal episodic GH secretory pattern returned. Intravenous infusions of insulin which reduced, but did not completely normalize, blood glucose levels, allowed the resumption of growth and pulsatile GH secretion. Continuous infusion of recombinant human insulin-like growth factor-I (hIGF-I) at 1110 pmol/h for 54 h also caused a large initial rise in body weight in diabetic rats (17·1 ± 1·6 compared with 7·5 ± 2·8 g in saline-infused controls) due primarily to increased fluid retention. This effect of hIGF-I occurred without any significant changes in pituitary GH, AVP, oxytocin, blood glucose or bone growth over this short-term infusion, nor was there any obvious effect on spontaneous GH secretion, monitored over the entire infusion period. We conclude that the diabetic rat is not a good model to study growth stimulation by short-term insulin or IGF-I treatments because the insulin-like effects of these peptides obscure their specific growth-promoting activities in this model.
Journal of Endocrinology (1989) 122, 661–670
SA Hinke, RP Pauly, J Ehses, P Kerridge, HU Demuth, CH McIntosh, and RA Pederson
It is well documented that the release of insulin from isolated perifused islets attenuates over time, despite a continued glucose stimulation. In the current study we have shown that potentiation of insulin release by the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP) is also attenuated after its continuous application. In less than 20 h of maintained stimulus with either hyperglycaemia (11.0 mM glucose) or GIP (10 nM) under hyperglycaemic conditions, insulin release returned to basal values. This was not due to loss of islet viability or reduction in the releasable pool of insulin granules, as 1 mM isobutylmethylxanthine was able to stimulate equivalent insulin release under both conditions. Further examination of chronic GIP desensitization was examined in cultured mouse insulinoma (betaTC-3) cells. GIP-stimulated cAMP production was not greatly affected by the prevailing glucose conditions, suggesting that the glucose dependence of GIP-stimulated insulin release occurs distally to the increase in intracellular cAMP in betaTC-3 cells. The GIP-stimulated cAMP response curve after desensitization was of similar magnitude at all glucose concentrations, but GIP pretreatment did not affect forskolin-stimulated cAMP production. Desensitization of the cAMP response in betaTC-3 cells was shown not to involve induction of dipeptidyl peptidase IV or pertussis toxin-sensitive G-proteins, activation of protein kinase C or protein kinase A, or modulation of phosphodiesterase activity. Homologous desensitization of the insulin-potentiating activity of GIP was found to affect both GIP-stimulated and forskolin-stimulated insulin release, indicating desensitization of distal steps in the stimulus-exocytosis cascade.
C Stewart, N A Taylor, I C Green, K Docherty, and C J Bailey
Insulin delivery by somatic cell gene therapy was evaluated using murine pituitary AtT20MtIns-1.4 cells. These cells have been stably transfected to release human insulin by the introduction of a recombinant plasmid bearing a human preproinsulin cDNA under the control of a zinc-sensitive metallothionein promoter. 6 × 107 AtT20MtIns-1.4 cells were implanted subcutaneously into streptozotocin-diabetic mice immunosuppressed with cyclosporin A. Release of human insulin was assessed using a specific plasma human C-peptide assay. On days 1 and 2 after implantation human C-peptide concentrations were about 0·02 pmol/ml. Consumption of zinc sulphate solution (500 mg/l) as drinking fluid for days 3–5 increased plasma human C-peptide concentrations to 0·11 ±0·01 pmol/ml (mean±s.e.m.), n=11, P<0·01, and concentrations declined when zinc was discontinued. The extent of hyperglycaemia was slightly lower (P<0·05) than in a group implanted with non-transfected AtT20 cells. The study was terminated after 9 days, and tumour-like aggregations of implanted cells were identified at autopsy. These comprised a large necrotic core with insulin-containing cells at the periphery. The study provides support for the view that somatic cell gene therapy offers a potential approach to insulin delivery in diabetes mellitus.
Journal of Endocrinology (1994) 142, 339–343
M. C. Neville, V. S. Sawicki, and W. W. Hay Jr
In order to determine whether short term variations in plasma glucose and/or insulin influence milk lactose secretion in women, the effects of fasting and increased blood insulin and glucose on milk volume and composition were studied with glucose clamp methodology in exclusively and partially breast-feeding women. Twenty hours of fasting had no discernable effect on the output of milk or its macronutrient composition. Four hours of increased plasma insulin, studied under conditions where plasma glucose was maintained at the fasting level, had no effect on milk volume, milk glucose concentration, total fat content or lactose secretion rate. Increased plasma glucose, maintained at twice fasting levels for 4 to 6 h, produced a threefold increase in milk glucose concentration but had no significant effect on the rate of lactose synthesis. In partially breast-feeding women producing no more than 200 ml milk per day, a similar degree of hyperglycaemia increased milk glucose more than fourfold but did not significantly increase the milk secretion rate. It is concluded that human milk production is isolated from the homeostatic mechanisms that regulate glucose metabolism in the rest of the body, in part because the lactose synthetase system has a K m for glucose lower than the concentration available in the Golgi compartment.
Journal of Endocrinology (1993) 139, 165–173
Intravenous injection of 8·5 nmol (1–24)ACTH increased the plasma levels of glucagon, insulin, glucose and free fatty acids in rabbits. The (1–24)ACTH-induced hyperglucagonaemia and hyperinsulinaemia started 3 and 20 min after the injection respectively. Similar increases in the plasma levels of glucagon, insulin and free fatty acids were found with 5·3 nmol (1–39)ACTH, whereas (1–4)ACTH, (4–10)ACTH, (1–10)ACTH, (11–24)ACTH, (7–38)ACTH and (18–39)ACTH (corticotrophin-like intermediate lobe peptide) injected at doses of approximately 8 nmol were inactive. Infusions with the alpha-adrenergic blocking drug, phentolamine, reduced the (1–24) ACTH-induced hyperglucagonaemia and hypergly-caemia, and augmented the (1–24)ACTH-induced hyperinsulinaemia, which now became significant after 5 min. Infusions with the beta-adrenergic blocking drug, propranolol, did not diminish the (1–24)ACTH-induced effects, but killed the rabbits after 2–4 h.
It is concluded that the acute in-vivo effects of ACTH in rabbits are modulated by the involvement of alpha-adrenergic receptors, which increased the plasma levels of glucagon and glucose, and delayed and diminished the ACTH-induced increases in the plasma levels of insulin. The (1–24)ACTH-induced increases in the plasma levels of free fatty acids were not influenced by the adrenergic blocking drugs.
J. Endocr. (1984) 100, 345–352
R. N. BARTON and R. A. LITTLE
In rats injected with a specific inhibitor of adrenal steroidogenesis, 4α,5-epoxy-17β-hydroxy-3-oxo-5α-androstane-2α-carbonitrile (WIN 24 540), the increase in the concentration of corticosterone in the plasma caused by bilateral hind-limb ischaemia was greatly reduced. The lethality of this injury was increased. Fluid loss into the hind limbs was unaltered but the compensatory movement of fluid into the plasma was impaired. The hyperglycaemia that occurred after the injury was markedly reduced by WIN 24 540, but this was probably unrelated to the poorer compensation of fluid loss as there was no corresponding fall in plasma osmolality. The effects of injecting WIN 24 540 could be completely reversed by the simultaneous injection of corticosterone, so as to restore the rise in its concentration in the plasma. We conclude that the adrenal cortex plays an active part in the early circulatory and metabolic responses to injury, in contrast to its reportedly permissive role in the later catabolic phase.
I. G. WHITE
1. The effect of twice-daily injections of formalin, as an agent eliciting the adaptation syndrome, has been studied on the blood constituents of sheep.
2. Within 4 days of the start of injections of 5 ml. of 4 % formalin there was a significant hypochloraemia, hypoglycaemia and leucocytosis with a return to normal levels after 10 days. There was also an intermediate compensatory hyperglycaemia. The leucocytosis was due primarily to a neutrophilia, although there was a slight transient increase in both absolute lymphocyte and eosinophil counts.
3. When the dose rate was increased to 10–20 ml. of 10% formalin, the plasma chloride and leucocyte patterns were repeated and there was also an increase in erythrocytes and haemoglobin values. In the later stages of the experiment the absolute lymphocyte and eosinophil counts were depressed, whilst the absolute monocyte count had risen.
4. There were no significant changes in blood acetone or in the basophil count throughout the experiment.
5. It is concluded that, in general, the blood changes elicited by an alarming stimulus in sheep are similar to those described in small laboratory animals. The renewed response to an increased intensity of stimulus observed in this experiment indicates that resistance is quantitatively as well as qualitatively specific.
P. E. B. REILLY and E. J. H. FORD
Continuous infusions of tracer amounts of [U-14C]glucose were used to measure the effect of a single intramuscular injection of 0·5 mg betamethasone/kg on the rates of glucose entry in six sheep eating 900 g/day chopped hay containing 49 g protein. In four of the sheep the rates of oxidation of glucose to CO2 were also measured. Hyperglycaemia occurred with a maximum plasma glucose concentration occurring 24 h after administration of the steroid. The mean glucose entry rates, expressed as mg/min/kg ± s.e.m., rose from 1·27 ± 0·11 (6) to 1·75 ± 0·16 (6). The difference between these means is significant (P < 0·001). The mean proportion of CO2 derived from glucose after administration of betamethasone (11·64 ± 1·4% (4)) was not significantly different (P > 0·5) from that derived before its administration (12·47 ± 0·34% (4)). The amounts of glucose available for synthetic purposes increased because the total production rates increased. The mean extra amount available was 11·3 ± 2·1 (4) mg C/min. These results are discussed in the light of other findings relating to the actions of the glucocorticosteroids on glucose metabolism.
R. L. REID, N. T. HINKS, and S. C. MILLS
Diabetes, uncomplicated by severe renal damage, was established in ewes, 115–135 days pregnant, by injection of 40 mg. alloxan/kg. body weight. The onset of severe hyperketonaemia was accompanied by loss of appetite and rapid loss of body weight, death ensuing usually after 10–16 days. Blood acetate levels were markedly elevated in the early stages of the syndrome, and declined as feed intake fell. It is concluded that the high levels are a consequence of a block in the metabolism of acetate absorbed from the gut. Levels remained above normal after complete loss of appetite, to a degree correlated with blood ketone level. Fasting blood glucose levels were reduced in pregnant ewes below those of non-pregnant ewes, to a degree which correlated with foetal weight. Hypoglycaemia was recorded in fasting diabetic ewes with triplets. Plasma cortisol levels were only slightly increased after alloxan, except in pre-mortal stages, when very high values were associated with terminal increases in blood glucose, NPN and creatinine levels.
A dose of 20 mg. alloxan/kg. temporarily increased blood glucose levels and induced a transitory diabetes in two of twelve ewes. Hyperglycaemia and mild ketosis occurred, but were not associated with loss of appetite or loss of body weight. A dose of 60 mg./kg. induced a diabetic-uraemic syndrome in three of eight ewes, characterized by gross elevation of glucose, NPN, creatinine, amino-N and cortisol levels. This syndrome occurred in only two of sixteen ewes after 40 mg. alloxan/kg.