Thiazolidinedione insulin sensitizers and the heart: a tale of two organs?

Thiazolidinediones (TZDs) are relatively new agents for the treatment of type 2 diabetes. They act as agonists at the PPAR-gamma nuclear receptor and their therapeutic effects include decreased insulin resistance and hyperglycaemia, an improved plasma lipid, inflammation and pro-coagulant profile, and amelioration of hypertension, microalbuminuria and hepatic steatosis. The most common side effects of TZDs include weight gain and oedema, with occasional reports of congestive heart failure (CHF). This review discusses the benefit-risk profile of TZDs in treating patients with type 2 diabetes, with particular reference to the heart. To provide context, we explore briefly the epidemiology and pathophysiology of heart failure in patients with type 2 diabetes, touch on the association of heart disease and cardiovascular mortality with antihyperglycaemic treatment modalities other than TZDs, and then focus on the effects of TZDs on the heart, cardiovascular risk factors and outcomes. We describe the cluster of host factors, which seems to predispose patients with type 2 diabetes to TZD-induced or TZD-exacerbated oedema and CHF and then provide an overview of the putative mechanisms of these TZD-related side effects. We also propose that certain diuretics (amiloride and spironolactone), by targeting the distal nephron that expresses PPARgamma in collecting duct cells, might be of benefit in ameliorating the fluid retention and oedema associated with TZDs.

Energy restriction enhances therapeutic efficacy of the PPARgamma agonist, rosiglitazone, through regulation of visceral fat gene expression.

AIM: Consumption of a palatable diet can induce hyperphagia, leading to weight gain (dietary obesity) and insulin resistance in rats. Thiazolidinediones (TZDs) can also induce hyperphagia in rats but conversely have an insulin-sensitizing effect. The aim of this study was to investigate whether preventing TZD-induced hyperphagia (i.e. energy restriction) in dietary obese (DIO) rats would enhance the insulin-sensitizing effects of treatment at a therapeutic dose; and, within this paradigm, to produce an original survey of candidate TZD-gene targets in the clinically relevant visceral white adipose tissue (WAT) depot. METHODS: DIO rats that were either freely fed or energy restricted (i.e. pair-fed to the level of untreated controls) were treated with rosiglitazone maleate (RSG; 3 mg/kg/day) for 2 weeks, the restricted group controlling for treatment-induced hyperphagia and weight gain. The outcome measures were circulating concentrations of various biochemical markers of insulin resistance, and gene expression was measured in epididymal WAT. RESULTS: In both freely fed and pair-fed groups, compared to untreated DIO controls, RSG reduced plasma levels of insulin (-29% and -43%; p < 0.05 and p < 0.001, respectively), free fatty acids (FFAs; -45% and -48%; p < 0.01 and p < 0.001, respectively) and triglycerides (TGs; -63% and -72%; both p < 0.001), reflected in improved insulin sensitivity, as measured by homeostasis model assessment (-29% and -43%; p < 0.01 and p < 0.0001). RSG also increased the expression of the fatty acid transport/synthesis genes, fatty acid transport protein (2.4-3.2-fold), epidermal fatty acid-binding protein (FABP; 1.7-2.0-fold), heart FABP (25-29-fold) and fatty acid synthase (2.3-2.9-fold; all p < 0.05) in both groups. Adipocyte FABP was also increased by RSG treatment, but only in combination with energy restriction (1.52-fold; p < 0.05) as was hexokinase II expression (p < 0.001). In contrast, the drug had no effect on expression of several genes associated with lipolysis. Although obesity-induced hyperleptinaemia was normalized only in the energy-restricted group, leptin messenger RNA (mRNA) expression was reduced in both treated groups (all p < 0.01). Resistin and tumour necrosis factor-alpha expression was also reduced, though in the latter case, only with energy restriction (p < 0.05). Other adipokines were unaffected by RSG treatment. CONCLUSION: Our results clearly show that energy restriction enhances the therapeutic efficacy of TZDs and suggest that this occurs, at least in part, through a modulatory effect on gene expression in visceral WAT. These findings improve our understanding of the underlying mechanistic basis for the clinical usefulness of dietary restriction as an adjunct to TZD therapy in type 2 diabetes.

Rapid reversal of hepatic steatosis, and reduction of muscle triglyceride, by rosiglitazone: MRI/S studies in Zucker fatty rats.

AIM: This study aimed to chart the time course and durability of the effects of rosiglitazone, a potent thiazolidinedione-based peroxisome proliferator-activated receptor gamma agonist, on hepatic steatosis and intramyocellular lipid in an animal model of obesity, the Zucker Fatty (ZF) rat. METHODS AND RESULTS: Rosiglitazone (3 mg/kg/day p.o.) significantly reduced both liver fat content (by 59%; p < 0.05) and size (11.5%; p < 0.05) in male ZF rats that received between 3 days and 1 week of treatment, and these reductions were maintained for at least 12 weeks. Liver fat content measured by magnetic resonance spectroscopy (MRS) correlated closely and positively with plasma insulin levels (reduced by 89% within a week, r = 0.8) and with postmortem histological fat fractional volume (r = 0.89). Similarly, liver volume measured by magnetic resonance imaging (MRI) correlated closely with postmortem wet weight (r = 0.99). MRS also showed, and numbers of lipid vacuoles counted in transmission electron micrographs confirmed, that rosiglitazone significantly reduced the elevated intramyocellular lipid seen in ZF rat skeletal muscle by at least 40% (p < 0.05). CONCLUSIONS: Localized MRS and MRI showed that rosiglitazone reversed the hepatic steatosis, hepatomegaly and intramyocellular lipid, characteristic of the ZF rat, an animal model of obesity.

In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

Insulin-sensitizing action of rosiglitazone is enhanced by preventing hyperphagia.

AIM: We investigated whether pair-feeding to prevent hyperphagia would potentiate the insulin-sensitizing effect of rosiglitazone in chow-fed and insulin-resistant dietary obese rats, and studied the role of leptin and hypothalamic neuropeptide Y as mediators of weight gain during treatment. METHODS: Dietary obese and chow-fed rats (575 +/- 10 vs. 536 +/- 7 g; p < 0.01) were given rosiglitazone (30 mg/kg p.o.) or vehicle daily for 14 days. RESULTS: Energy intake and weight gain were greater in rosiglitazone-treated ad-lib-fed rats (body weight: chow + 24 +/- 2 g, rosiglitazone-treated + 55 +/- 2 g, p < 0.001; dietary obese + 34 +/- 2 g, rosiglitazone-treated + 74 +/- 7 g, p < 0.001). Half of each rosiglitazone-treated group were pair-fed to vehicle-treated controls. Rosiglitazone normalized circulating free fatty acids (FFAs) and insulin sensitivity in dietary obese rats (homeostasis model assessment (HOMA): chow-fed controls, 3.9 +/- 0.3; dietary obese controls, 6.7 +/- 0.7; rosiglitazone-treated, ad lib-fed dietary obese, 4.2 +/- 0.5; both p < 0.01). Insulin sensitivity improved further with pair-feeding (HOMA: 2.9 +/- 0.4; p < 0.05 vs. rosiglitazone-treated, ad lib-fed dietary obese), despite unchanged FFAs. Qualitatively similar findings were made in chow-fed rats. Pair-feeding prevented rosiglitazone-related weight gain in chow-fed, but not dietary obese rats (body weight: + 49 +/- 5 g, p < 0.001 vs. untreated dietary obese controls). Adipose tissue OB mRNA was elevated in dietary obese rats, reduced 49% (p < 0.01) by rosiglitazone treatment, and further (by 16%) with pair-feeding (p < 0.0001). Plasma leptin, however, only fell in the pair-fed group. Hypothalamic neuropeptide Y mRNA was unchanged throughout, suggesting that weight gain associated with high-dose rosiglitazone treatment is independent of hypothalamic neuropeptide Y. CONCLUSIONS: Food restriction potentiates the insulin-sensitizing effect of rosiglitazone in rats, and this effect is independent of a fall in FFAs.

Beta-cell mass dynamics in Zucker diabetic fatty rats. Rosiglitazone prevents the rise in net cell death.

The evolution of diabetes in the male leptin receptor-deficient (fa/fa) Zucker diabetic fatty (ZDF) rat is associated with disruption of normal islet architecture, beta-cell degranulation, and increased beta-cell death. It is unknown whether these changes precede or develop as a result of the increasing plasma glucose, or whether the increased beta-cell death can be prevented. Early intervention with thiazolidinediones prevents disruption of the islet architecture. To determine the specific effects of rosiglitazone (RSG) on beta-cell mass dynamics, male fa/fa (obese) and +/fa or +/+ (lean) rats age 6 weeks were fed either chow (control group [CN]) or chow mixed with rosiglitazone (RSG group) at a dosage of 10 micromol. kg(-1) body wt.day(-1). Rats were killed after 0, 2, 4, 6, or 10 weeks of treatment (at age 6, 8, 10, 12, or 16 weeks). Plasma glucose increased from 8.9 +/- 0.4 mmol/l at 0 weeks to 34.2 +/- 1.8 mmol/l (P = 0.0001) at 6 weeks of treatment in obese CN rats and fell from 8.0 +/- 0.3 to 6.3 +/- 0.4 mmol/l in obese RSG rats (P = 0.02). beta-cell mass fell by 51% from 2 to 6 weeks of treatment (ages 8-12 weeks) in obese CN rats (6.9 +/- 0.9 to 3.4 +/- 0.5 mg; P < 0.05), whereas beta-cell mass was unchanged in obese RSG rats. At 10 weeks of treatment (age 16 weeks), beta-cell mass in obese CN rats was only 56% of that of obese RSG rats (4.4 +/- 0.4 vs. 7.8 +/- 0.3 mg, respectively; P = 0.0001). The beta-cell replication rate fell from a baseline value of 0.95 +/- 0.12% in lean rats and 0.94 +/- 0.07% in obese rats (at 0 weeks) to approximately 0.3-0.5% in all groups by 6 weeks of treatment (age 12 weeks). After 10 weeks of treatment, beta-cell replication was higher in obese RSG rats than in CN rats (0.59 +/- 0.14 vs. 0.28 +/- 0.05%, respectively; P < 0.02). Application of our mass balance model of beta-cell turnover indicated that net beta-cell death was fivefold higher in obese CN rats as compared with RSG rats after 6 weeks of treatment (age 12 weeks). The increase in beta-cell death in obese CN rats during the 6-week observation period was well correlated with the increase in plasma glucose (r2 = 0.90, P < 0.0001). These results suggest that the development of hyperglycemia in ZDF rats is concomitant with increasing net beta-cell death. beta-cell proliferation compensates for the increased beta-cell loss at a time when plasma glucose is moderately elevated, but compensation ultimately fails and the plasma glucose levels increase beyond approximately 20 mmol/l. Treatment with rosiglitazone, previously shown to reduce insulin resistance, prevents the loss of beta-cell mass in obese ZDF rats by maintaining beta-cell proliferation and preventing increased net beta-cell death.

Effects of chronic murine and human leptin infusion on plasma leptin and corticosterone levels and energy balance in lean Zucker rats.

AIM: To clarify whether centrally delivered leptin can access the circulation and to determine to what extent the effects of i.c.v. h-leptin and m-leptin on body weight and plasma corticosterone are due to reduced food intake. METHODS: Male lean Zucker rats were infused i.c.v. with recombinant m-leptin or h-leptin (42 microg/day) for 7 days. Terminal plasma leptin levels were measured using selective r-leptin, m-leptin and h-leptin RIA. Plasma h-leptin and corticosterone levels were determined on days 0, 2, 4 and 6 of h-leptin infusion. Interscapular brown adipose tissue weight and UCP-1 mRNA expression (an indicator of thermogenic capacity) were also measured. RESULTS: The terminal plasma leptin level was elevated (from 2.2 +/- 0.4 to 42.7 +/- 20.2 ng/ml) in the h-leptin-treated lean rats to levels similar to those in vehicle i.c.v. infused fa/fa rats (72.2 +/- 4.7 ng/ml), but this was only detectable when the h-leptin radioimmunoabsorbent assay (RIA) was used. Further, both m-leptin and h-leptin infusions in lean rats elevated terminal plasma corticosterone (352 +/- 37 and 389 +/- 55 ng/ml, respectively) to levels similar to those in i.c.v. rats (386 +/- 62 ng/ml), whereas diet-restriction by pair-feeding, with the h-leptin group, in lean rats had no effect (207 +/- 45 ng/ml). The increase in plasma corticosterone level coincided with the maximum hypophagic effects of leptin and preceded the appearance and sustained elevation of exogenous human leptin in the circulation. Both m-leptin and h-leptin i.c.v. infusion reduced body weight gain (3% and 4%, respectively, compared to pair-fed group) and increased UCP-1 expression (11-fold and 16-fold, respectively) in lean rats. However, h-leptin elicited an earlier effect than m-leptin on body weight, manifested as an earlier reduction in food intake and greater increase in UCP-1 expression. h-Leptin also elicited a greater reduction in body weight gain than did pair-feeding. CONCLUSIONS: Intracerebroventricular-infused m-leptin or h-leptin was detected in the circulation. Furthermore, m-leptin and h-leptin elevated plasma corticosterone levels and h-leptin caused some weight loss in lean rats independently of its suppression of food intake. The elevation of corticosterone levels in the lean rats may be a mechanism whereby they resist excessive weight loss in response to leptin.

Central exendin-4 infusion reduces body weight without altering plasma leptin in (fa/fa) Zucker rats.

OBJECTIVE: To investigate whether chronic administration of the long-acting glucagon-like peptide-1 receptor agonist exendin-4 can elicit sustained reductions in food intake and body weight and whether its actions require an intact leptin system. RESEARCH METHODS AND PROCEDURES: Male lean and obese Zucker (fa/fa) rats were infused intracerebroventricularly with exendin-4 using osmotic minipumps for 8 days. RESULTS: Exendin-4 reduced body weight in both lean and obese Zucker rats, maximum suppression being reached on Day 5 in obese (8%) and Day 7 in lean (16%) rats. However, epididymal white adipose tissue weight was not reduced, and only in lean rats was there a reduction in plasma leptin concentration. Food intake was maximally suppressed (by 81%) on Day 3 in obese rats but was reduced by only 18% on Day 8. Similarly, in lean rats food intake was maximally reduced (by 93%) on Day 4 of treatment and by 45% on Day 8. Brown adipose tissue temperature was reduced from Days 2 to 4. Plasma corticosterone was elevated by 76% in lean but by only 28% in obese rats. DISCUSSION: Chronic exendin-4 treatment reduced body weight in both obese and lean Zucker rats by reducing food intake: metabolic rate was apparently suppressed. These effects did not require an intact leptin system. Neither does the absence of an intact leptin system sensitize animals to exendin-4. Partial tolerance to the anorectic effect of exendin-4 in lean rats may have been due to elevated plasma corticosterone and depressed plasma leptin levels, but other counter-regulatory mechanisms seem to play a role in obese Zucker rats.

Down-regulation of orexin gene expression by severe obesity in the rats: studies in Zucker fatty and zucker diabetic fatty rats and effects of rosiglitazone.

Orexins (hypocretins) are lateral hypothalamic neuropeptides implicated in regulating feeding and the sleep-wake cycle. To study their possible relevance to obesity and diabetes, we measured hypothalamic prepro-orexin mRNA levels in obese, normoglycemic Zucker fatty (fa/fa) and in hyperglycemic, non-obese Zucker diabetic fatty (ZDF) rats. Hypothalamic prepro-orexin mRNA concentrations in Zucker fatty rats were 31% lower than those in lean controls (0. 69+/-0.06 vs. 1.00+/-0.10 arbitrary units, P<0.05), but did not differ between ZDF diabetic rats and non-diabetic controls. Treatment of ZDF diabetic rats with rosiglitazone (1 or 3 mg/kg body weight daily for 13 weeks) normalized plasma glucose and significantly reduced plasma insulin, while leptin levels were 67% higher than in untreated ZDF rats (20.2+/-0.5 vs. 12.1+/-2.5, P<0. 001). Rosiglitazone treatment markedly enhanced weight gain compared with untreated ZDF rats (final weight 732+/-13 g vs. 409+/-13 g, P<0. 001) even though they were restricted to the same food intake. Rosiglitazone-treated ZDF rats had significantly lower hypothalamic prepro-orexin mRNA levels (0.68+/-0.07 arbitrary units) than both non-diabetic lean controls (1.00+/-0.10, P=0.02) and untreated diabetics (1.03+/-0.14, P=0.03). Our data suggest that prepro-orexin gene expression may be suppressed by substantial weight gain. Obesity-related signals that might mediate this effect have not been identified, but plasma leptin, insulin and glucose are not obviously involved.

Rosiglitazone prevents the onset of hyperglycaemia and proteinuria in the Zucker diabetic fatty rat.

AIM: To investigate the potential of rosiglitazone, a highly potent agonist at the nuclear peroxisome proliferator activated receptor-gamma (PPAR-gamma), to prevent the development of diabetes in the Zucker diabetic fatty (ZDF) rat or to ameliorate the condition at a later stage of the disease. METHODS: Rosiglitazone (10 micromol/kg body weight daily) was given via the diet to ZDF rats from aged 6 weeks, before the onset of hyperglycaemia (Prevention group), or from aged 21 weeks after hyperglycaemia and proteinuria were established (Intervention group). Untreated ZDF rats and age-matched Zucker lean rats (ZL) served as controls and the experiment was terminated when the animals were aged 28 weeks. RESULTS: Whilst the combined ZDF control and Intervention groups were already hyperglycaemic (14.6 +/- 1.6 vs. ZL 5.7 +/- 0.1 mmol/l, mean +/- s.e.m.; p < 0.05), glycosuric and polydipsic at aged 11 weeks, and thereafter had a declining plasma insulin concentration, rosiglitazone Prevention treatment maintained normoglycaemia even at aged 27 weeks (3.7 +/- 0.3 mmol/l vs. ZL 3.0 +/- 0.3 mmol/l; NS). Intervention treatment at aged 21 weeks, however, failed to ameliorate the diabetes. These functional data were supported by determinations of pancreatic insulin content (microg/mg tissue as follows: ZL, 43.1 +/- 3.9; ZDF control (28 weeks) + ZDF Intervention control (21 weeks), 6.0 +/- 0.8; Prevention, 63.6 +/- 15.8; Intervention, 6.2 +/- 0.9) and by morphological, immunohistochemical and electron microscopical examination of pancreata at the end of the study. Thus, islets from rosiglitazone Prevention rats were similar to ZL rats, whereas ZDF controls and Intervention rats exhibited islets depleted of insulin, with a disorganized architecture and an ultrastructure indicative of work hypertrophy. ZDF control rats and Intervention rats, though not rosiglitazone Prevention rats, also exhibited marked proteinuria, indicative of renal glomerular damage. CONCLUSIONS: Our results demonstrate that in ZDF rats, rosiglitazone prevents the progression from insulin resistance to overt diabetes. These data provide a rationale for investigating whether treatment with rosiglitazone of patients with early signs of perturbed glucose metabolism (e.g. impaired fasting glucose (IGT)) may prevent the progression to type 2 diabetes and its associated complications.

Therapeutic index for rosiglitazone in dietary obese rats: separation of efficacy and haemodilution.

1. The blood glucose-lowering efficacy of rosiglitazone (RSG) and the mechanisms of associated weight gain were determined in dietary obese rats (DIOs). DIO and chow-fed rats received RSG 0.3-30 mg kg-1 daily for 21 days. 2. In DIOs, plasma glucose and insulin concentrations were reduced by RSG at dosages of 3 and 10 mg kg-1, respectively. Homeostasis model assessment (HOMA) indicated the threshold for a reduction of insulin resistance was 1 mg kg-1. Neither glucose nor insulin levels were affected by treatment in chow-fed rats. 3. RSG 0.3 mg kg-1 lowered free fatty acids (FFAs) in DIOs, whereas for plasma triglycerides (TGs), the threshold was 3 mg kg-1. By contrast, the threshold for reducing packed red cell volume (PCV) and increasing cardiac mass was 10 mg kg-1. Thus, the therapeutic index for RSG in DIOs was >3 and < or = 10. 4. Energy intake and weight gain increased in treated DIOs (by 20% and 50 g, at 30 mg kg-1) and chow-fed rats (by 25% and 35 g, at 30 mg kg-1). In DIOs, these increases coincided with falls in plasma leptin (40% lower at 30 mg kg-1) and insulin (43% lower at 30 mg kg-1). By contrast, in chow-fed rats, weight gain and hyperphagia occurred without changes in either leptin or insulin. However, reductions in FFAs below 0.4 - 0.3 mM were associated with hyperphagia and weight gain in DIO and chow-fed rats. 5. We conclude that increased energy intake and body weight did not attenuate the improved metabolism evoked by RSG in DIO rats, and that insulin action was enhanced at a dose >3 fold below the threshold for causing haemodilution and cardiac hypertrophy in DIO rats.

Differences between the vasorelaxant activity of adenosine-receptor agonists on guinea-pig isolated aorta precontracted with noradrenaline or phenylephrine.

The relaxant effect of adenosine and 5'-(N-ethylcarboxamido)adenosine (NECA) against alpha-adrenoceptor-mediated contractile tone in guinea-pig isolated aortic rings has been examined to determine if this A2B-receptor-mediated relaxation was dependent upon the contracting agent, and whether the contractions were dependent upon intracellular or extracellular calcium. Relaxation responses were consistently greater for aortic rings pre-contracted with phenylephrine (3x10(-6) M) than for rings pre-contracted with noradrenaline (3x10(-6) M). Maximum inhibition by NECA was significantly greater for phenylephrine-contracted aortae than for noradrenaline-contracted (81.9+/-2.8% compared with 25.0+/-1.5%). These differences persisted in the presence of beta- and alpha2-adrenoceptor blockade and could not, therefore, be attributed to stimulation of these receptors by noradrenaline. The ratio of the contractions obtained before and in the presence of adenosine or NECA was compared with the control ratio obtained before and after vehicle. Experiments were performed both in the presence of normal calcium levels and under calcium-free conditions. In normal-calcium medium, NECA inhibited phenylephrine-induced contractions (test ratio, 76.7+/-3.9%; control ratio, 133.1+/-9.8%) to a greater extent than noradrenaline-induced contractions (108.4+/-4.1 and 123.4+/-4.9%); adenosine similarly inhibited phenylephrine-induced contractions more than those induced by noradrenaline. Under calcium-free conditions, adenosine (36.7+/-11.9 and 110.7+/-26.6%) and NECA (55.2+/-9.1 and 87.1+/-14.9%) were only effective against phenylephrine-induced contractions. This suggests that activation of the A2B-receptor by these agonists inhibited intracellular mobilization of calcium for phenylephrine-induced contractions only. The effects on extracellular calcium influx were examined for phenylephrine- and noradrenaline-induced contractions in normal-calcium medium but in the presence of ryanodine to prevent intracellular calcium mobilization. NECA inhibited phenylephrine-induced contractions (77.3+/-12.4 and 111.4+/-9.3%), presumably by interfering with influx of calcium through receptor-operated calcium channels. In contrast, NECA failed to reduce noradrenaline-induced contractions (121.5+/-10.7 and 122.4+/-11.6%), suggesting that the effect on noradrenaline is predominantly via interaction with intracellular calcium. Adenosine was consistently a more effective relaxant than NECA, possibly because of an additional intracellular component of the response. We conclude that adenosine receptor agonists inhibit phenylephrine-induced contractions of guinea-pig aorta more selectively than noradrenaline-induced contractions. A2B-receptor stimulation might reveal a fundamental difference between the modes of contraction elicited by these two alpha-adrenoceptor agonists.

Hypothalamic orexin expression: modulation by blood glucose and feeding.

Orexins (hypocretins), novel peptides expressed in specific neurons of the lateral hypothalamic area (LHA), stimulate feeding when injected intracerebroventricularly. We investigated their role in feeding in the rat by measuring hypothalamic prepro-orexin mRNA levels under contrasting conditions of increased hunger. Prepro-orexin mRNA levels increased significantly after 48 h of fasting (by 90-170%; P < 0.05) and after acute (6 h) hypoglycemia when food was withheld (by 90%; P < 0.02). By contrast, levels were unchanged during chronic food restriction, streptozotocin-induced diabetes, hypoglycemia when food was available, voluntary overconsumption of palatable food, or glucoprivation induced by systemic 2-deoxy-D-glucose. Orexin expression was not obviously related to changes in body weight, insulin, or leptin, but was stimulated under conditions of low plasma glucose in the absence of food. Orexins may participate in the short-term regulation of energy homeostasis by initiating feeding in response to falls in glucose and terminating it after food ingestion. The LHA is known to contain neurons that are stimulated by falls in circulating glucose but inhibited by feeding-related signals from the viscera; orexin neurons may correspond to this neuronal population.

Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat.

Two novel hypothalamic neuropeptides, orexin-A and -B, are suggested to regulate feeding. A single intracerebroventricular injection of orexin-A (23.4 nmol), administered 3 h into the light phase, increased feeding in satiated rats and prolonged feeding in fasted rats; it also increased feeding when given 6 h into, but not at the start of, the dark phase. An 8-day intracerebroventricular infusion with orexin-A (18 nmol/day) increased daytime feeding on days 2 and 8, but nocturnal feeding was reduced and there was no change in 24 h intake. Orexin-B had no effects. These results demonstrate a circadian variation in feeding responses to orexin-A.

The thiazolidinedione rosiglitazone (BRL-49653) lowers blood pressure and protects against impairment of endothelial function in Zucker fatty rats.

Human obesity is associated with insulin resistance, hyperinsulinemia, and a predisposition to hypertension and vascular disease, the origin of which may lie in impairment of endothelial function. We tested the effects of the thiazolidinedione rosiglitazone on blood pressure and endothelial function in insulin-resistant fatty Zucker rats, which display hypertension and abnormal endothelial cell function. We studied fatty Zucker rats given rosiglitazone maleate (50 micromol/kg diet; n = 8) for 9-12 weeks (treated fatty), untreated fatty rats (n = 8), and lean rats (n = 8) given diet alone. At the end of the study, systolic blood pressure was significantly higher in untreated fatty (147 +/- 5 mmHg) than in lean rats (125 +/- 2 mmHg; P < 0.05), but rosiglitazone treatment prevented the development of hypertension in fatty rats (123 +/- 1 mmHg). Fasting hyperinsulinemia in untreated fatty rats (28.7 +/- 6.0 ng/ml) was significantly lowered by rosiglitazone (7.0 +/- 1.4 ng/ml; P < 0.05 vs. untreated fatty), but remained significantly higher than the levels seen in lean rats (1.5 +/- 0.4 ng/ml; P < 0.01). Mesenteric arteries were studied in a myograph. Maximal acetylcholine chloride (1.1 micromol/l)-induced relaxation of norepinephrine hydrochloride (NE)-induced constriction was impaired in untreated fatty (62.4 +/- 3.4%) vs. lean (74.3 +/- 3.5%; P = 0.01) rats; this defect was partially prevented by rosiglitazone (66.5 +/- 3.0%; P = 0.01 vs. untreated fatty). Insulin (50 mU/l) significantly attenuated the contractile response to NE in lean rats (14.7 +/- 3.3%; P = 0.02); this vasodilator effect of insulin was absent in untreated fatty rats at concentrations of 50-5,000 mU/l, but was partially restored by rosiglitazone (9.7 +/- 2.5% attenuation; P = 0.02 vs. no insulin). Thus, rosiglitazone prevents the development of hypertension and partially protects against impaired endothelial function associated with insulin resistance. These latter effects may contribute to the drug's antihypertensive properties.

Differential distribution of orexin-A and orexin-B immunoreactivity in the rat brain and spinal cord.

The orexins are recently identified appetite-stimulating hypothalamic peptides. We used immunohistochemistry to map orexin-A and orexin-B immunoreactivity in rat brain, spinal cord, and some peripheral tissues. Orexin-A- and orexin-B-immunoreactive cell bodies were confined to the lateral hypothalamic area and perifornical nuclei. Orexin-A-immunoreactive fibers were densely distributed in the hypothalamus, septum, thalamus, locus coeruleus, spinal cord, and near the ventricles, but absent from peripheral sites investigated. In contrast, orexin-B-immunoreactive fibers were distributed sparsely in the hypothalamus. Orexin cells are strategically sited to contribute to feeding regulation, but their widespread projections suggest that orexins have other physiological roles.

Characterisation of the neuropeptide Y receptor that mediates feeding in the rat: a role for the Y5 receptor?

Food intake was measured in freely fed rats following intracerebroventricular administration of neuropeptide Y (NPY) and several of its analogues and antagonists to investigate the hypothesis that the NPY Y5 receptor mediates feeding. Rat NPY (rNPY), rNPY(2-36) and rNPY(3-36) produced similar feeding responses over the dose range 0.7-7.0 nmol. Rat peptide YY (rPYY) was more potent and at least as efficacious as rNPY. [Leu31 Pro34]-rNPY (agonist potency: Y1 > Y5 > Y4 = y6) and human pancreatic polypeptide (hPP) produced flatter dose-response curves, suggesting partial agonism at the receptor(s). rNPY(13-36) (agonist potency: Y2 > Y5) had little activity and rPP was inactive. [D-Trp32]-NPY was a weak orexigenic agent given alone and, consistent with partial agonism, it markedly antagonised the response to porcine NPY (pNPY). Similarly, the receptor antagonist (Y1 > Y4) 1229U91 stimulated feeding slightly, and markedly inhibited rNPY-induced feeding. In contrast to a previous report, BIBP 3226 (70 nmol), another Y1 receptor antagonist, failed to inhibit the response to rNPY. Our data in vivo are inconsistent with findings that hPP, [Leu31 Pro34]-rNPY and [D-Trp32]-rNPY are full agonists at the rat cloned Y5 receptor. Thus, whilst the Y5 receptor may be involved, its participation as the sole receptor mediating the orexigenic action of NPY in the rat remains unproven.

Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats.

Rosiglitazone (BRL 49653), a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist and potent insulin action-enhancing agent, was given in the diet (50 micromol/kg of diet) to male Zucker rats ages 6-7 weeks for 9 months (prevention group). In this treatment mode, rosiglitazone prolonged the time to onset of proteinuria from 3 to 6 months and markedly reduced the rate of its subsequent progression. Progression was also retarded when treatment was commenced (intervention group) after proteinuria had become established (4 months; ages 24-25 weeks). In either treatment mode, rosiglitazone normalized urinary N-acetyl-beta-D-glucosaminidase activity, a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure that occurred coincidentally with the development of proteinuria in Zucker fatty control rats. The renal protective action of rosiglitazone was verified morphologically. Thus in the prevention group there was an absence of the various indexes of chronic nephropathy that were prominent in the Zucker fatty control group, namely, glomerulosclerosis, dilated tubules containing proteinaceous casts, a loss of functional microvilli on the tubular epithelium, and varying degrees of chronic interstitial nephritis. An intermediate pathology was observed in the intervention group. Also, pancreatic islet hyperplasia, ultrastructural evidence of beta-cell work hypertrophy, and derangement of alpha-cell distribution within the islet were prominent features of Zucker fatty control rats, but these adaptive changes were ameliorated (intervention group) or prevented (prevention group) by rosiglitazone treatment. These data demonstrate that treatment of Zucker fatty rats with rosiglitazone produced substantial protection over a prolonged period against the development and progression of renal injury and the adaptive changes to pancreatic islet morphology caused by sustained hyperinsulinemia.

Differential vasoactive effects of the insulin sensitizers rosiglitazone (BRL 49653) and troglitazone on human small arteries in vitro.

BRL 49653 (rosiglitazone) and troglitazone are thiazolidinedione insulin-sensitizing agents, which are undergoing clinical evaluation as treatments for NIDDM. Potential side effects of thiazolidinediones include edema and hemodilution. Although the underlying mechanisms are presently unclear, animal and human studies have demonstrated a vasodilator action of troglitazone, which could in theory cause fluid retention. This in vitro study compared the direct vasodilator effects of troglitazone and BRL 49653 in small arteries (n = 44) from human subcutaneous fat. In arterial rings with a functioning endothelium and preconstricted with norepinephrine (NE; 6 micromol/l), troglitazone (n = 22 vessels), but not BRL 49653 (1-100 micromol/l), caused a concentration-related relaxation (69.4 +/- 5.2% at 100 micromol/l; P < 0.01). In the presence of indomethacin (IM; 10 micromol/l; n = 12), this vasorelaxant effect of troglitazone was abolished (P < 0.01 vs. troglitazone alone) and replaced by enhanced vasoconstriction (58.5 +/- 39.5% over the NE baseline) similar in magnitude to that produced by troglitazone vehicle (ethanol) alone (n = 16; NS vs. ethanol vehicle). By contrast, BRL 49653 (100 micromol/l; n = 22) and an equivalent volume of ethanol alone (n = 12) caused similar degrees of vasoconstriction (18.7 +/- 14.6 and 22.5 +/- 8.0%, respectively; NS). In the presence of IM (10 micromol/l; n = 10), the vasoconstrictor effect of BRL 49653 was enhanced (41.5 +/- 14.4%), although not significantly (NS vs. BRL 49653 alone or ethanol alone). Additional studies in Wistar rat arteries showed a similar vasodilator effect of troglitazone that was not inhibited by L-NAME (100 micromol/l). The alpha-tocopherol moiety alone had no vasorelaxant effect at concentrations up to 300 micromol/l. Thus, in human arterial resistance vessels in vitro, BRL 49653 does not possess the direct, IM-sensitive vasorelaxant action of troglitazone. This vasodilation could, in theory, permit transmission of systemic pressure to the capillary bed.