George S. Drummond

Heme Oxygenase-1 Induction Remodels Adipose Tissue and Improves Insulin Sensitivity in Obesity-Induced Diabetic Rats

Obesity-associated inflammation causes insulin resistance. Obese adipose tissue displays hypertrophied adipocytes and increased expression of the cannabinoid-1 receptor. Cobalt protoporphyrin (CoPP) increases heme oxygenase-1 (HO-1) activity, increasing adiponectin and reducing inflammatory cytokines. We hypothesize that CoPP administration to Zucker diabetic fat (ZDF) rats would improve insulin sensitivity and remodel adipose tissue. Twelve-week-old Zucker lean and ZDF rats were divided into 4 groups: Zucker lean, Zucker lean–CoPP, ZDF, and ZDF–CoPP. Control groups received vehicle and treatment groups received CoPP (2 mg/kg body weight) once weekly for 6 weeks. Serum insulin levels and glucose response to insulin injection were measured. At 18 weeks of age, rats were euthanized, and aorta, kidney, and subcutaneous and visceral adipose tissues were harvested. HO-1 expression was measured by Western blot analysis and HO-1 activity by serum carbon monoxide content. Adipocyte size and cannabinoid-1 expression were measured. Adipose tissue volumes were determined using MRI. CoPP significantly increased HO-1 activity, phosphorylated AKT and phosphorylated AMP kinase, and serum adiponectin in ZDF rats. HO-1 induction improved hyperinsulinemia and insulin sensitivity in ZDF rats. Subcutaneous and visceral adipose tissue volumes were significantly decreased in ZDF rats. Adipocyte size and cannabinoid-1 expression were both significantly reduced in ZDF–CoPP rats in subcutaneous and visceral adipose tissues. This study demonstrates that HO-1 induction improves insulin sensitivity, downregulates the peripheral endocannabinoid system, reduces adipose tissue volume, and causes adipose tissue remodeling in a model of obesity-induced insulin resistance. These findings suggest HO-1 as a potential therapeutic target for obesity and its associated health risks.

L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1β and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1β and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.

Heme oxygenase: the key to renal function regulation

Heme oxygenase (HO) plays a critical role in attenuating the production of reactive oxygen species through its ability to degrade heme in an enzymatic process that leads to the production of equimolar amounts of carbon monoxide and biliverdin/bilirubin and the release of free iron. The present review examines the beneficial role of HO-1 (inducible form of HO) that is achieved by increased expression of this enzyme in renal tissue. The influence of the HO system on renal physiology, obesity, vascular dysfunction, and blood pressure regulation is reviewed, and the clinical potential of increased levels of HO-1 protein, HO activity, and HO-derived end products of heme degradation is discussed relative to renal disease. The use of pharmacological and genetic approaches to investigate the role of the HO system in the kidney is key to the development of therapeutic approaches to prevent the adverse effects that accrue due to an impairment in renal function.

Heme Oxygenase -1 Gene Therapy: Recent Advances and Therapeutic Applications

Heme oxygenase-1 (HO-1) is regarded as a sensitive and reliable indicator of cellular oxidative stress. Studies on carbon monoxide (CO) and bilirubin, two of the three (iron is the third) end products of heme degradation have improved the understanding of the protective role of HO against oxidative injury. CO is a vasoactive molecule and bilirubin is an antioxidant, and an increase in their production through an increase in HO activity assists other antioxidant systems in attenuating the overall production of reactive oxygen species (ROS), thus facilitating cellular resistance to oxidative injury. Gene transfer is used to insert specific genes into cells that are either otherwise deficient in or that underexpress the gene. Successful HO gene transfer requires two essential elements to produce functional HO activity. Firstly, the HO gene must be delivered in a safe vector, e.g., adenoviral, retroviral or leptosome based vectors, currently being used in clinical trials. Secondly, with the exception of HO gene delivery to either ocular or cardiovascular tissue via catheter-based delivery systems, HO delivery must be site and organ specific. This has been achieved in rabbit ocular tissues, rat liver, kidney and vasculature, SHR kidney, and endothelial cells [Abraham et al., 1995a; Abraham et al., 1995b; Abraham et al., 2002c; Quan et al., 2004; Sabaawy et al., 2000; Sabaawy et al., 2001; Yang et al., 2004]. In this review, we discuss the functional significance of the HO system in various pathophysiological conditions and the beneficial therapeutic applications of human HO gene transfer and gene therapy in a variety of clinical circumstances.

The Potent Heme Oxygenase Inducing Action of Arsenic and Parasiticidal Arsenicals

The administration of trivalent arsenic, either as sodium arsenite or as the trypanocidal drug melarsoprol, to rats produced a profound induction of microsomal heme oxygenase (EC 1.14.99.3) in both liver and kidney and a concomitant decrease in cytochrome P-450 content. In addition, perturbations of delta-aminolevulinate synthase were observed which showed an initial decline followed by a rebound increase in the activity of this enzyme with arsenical treatment. Pentavalent arsenic did not induce hepatic heme oxygenase but did induce the enzyme in kidney, although to a lesser extent (50%) than trivalent arsenic. Treatment of isolated chick embryo liver cells in vitro with sodium arsenite or the parasiticidal drug melarsoprol also showed a potent induction of heme oxygenase. These findings describe a new and potent ability of arsenic and parasiticidal arsenicals to induce heme oxygenase resulting in enhanced degradation of cellular heme.

Disposition of tin-protoporphyrin and suppression of hyperbilirubinemia in humans

Tin (Sn4+)‐Protoporphyrin, a potent competitive inhibitor of heme degradation to bile pigment, was cleared rapidly from plasma in normal subjects (t½~4 hours for plasma levels >5 nmol/ml, with evidence of dose‐dependent pharmacokinetics at lower plasma concentrations). Small amounts were excreted promptly in urine (0.1% to 5.6%) and more gradually in feces (3.7% to 11.3%). The only dose‐limiting (>1.0 μmol/kg, single dose) side effect was mild sensitivity to sunlight and long‐wave ultraviolet light. Absorption after intramuscular administration was rapid, but there was no absorption after oral dosing. In bile duct—ligated rats treated with Sn‐protoporphyrin, there was a substantial (approximately 50%) reduction in plasma bilirubin levels compared with levels in ligated control animals. Seven studies were carried out in four women with moderate to severe cholestasis secondary to primary biliary cirrhosis and in two men with Gilberts syndrome. In these studies Sn‐protoporphyrin (total doses of 0.25 to 2.0 μmol/ kg body weight) reduced plasma bilirubin levels to a varying degree (7% to 43%) promptly after its intravenous administration.

Sn-protoporphyrin inhibition of fetal and neonatal brain heme oxygenase. Transplacental passage of the metalloporphyrin and prenatal suppression of hyperbilirubinemia in the newborn animal.

Sn(tin)-protoporphyrin, a potent competitive inhibitor of heme oxygenase, can suppress hyperbilirubinemia in animal neonates and significantly reduce plasma bilirubin levels in animals and man. To further explore the biological actions and metabolic disposition of Sn-protoporphyrin, we have examined its effect in the suckling neonate when administered to the mother either 24-48 h before or immediately after birth. Sn-protoporphyrin, when administered before birth, crossed the placental membranes, inhibited fetal heme oxygenase, and suppressed the transient hyperbilirubinemia that occurs in the neonate after birth in a dose-dependent manner. Tissue heme oxygenase activity in the neonate was also lowered in a dose-dependent manner. The blood-brain barrier of the neonate was permeable to Sn-protoporphyrin for a period of between 20-28 d of postnatal life. Sn-protoporphyrin, however, was not retained in brain, but left the brain space with a t1/2 of 1.7 d. In addition, Sn-protoporphyrin administered once at birth to neonates inhibited brain heme oxygenase in a dose-dependent manner. The results of this study demonstrate that Sn-protoporphyrin can cross the placental membranes, inhibit tissue heme oxygenase activity in the fetus, and can also, following such prenatal treatment, suppress the hyperbilirubinemia of the newborn animal.

Direct in vitro effects of bis (tri-n-butyltin)oxide on hepatic cytochrome P-450

Bis(tri-n-butyltin)oxide, an agriculturally important biocidal agent, when added in vitro to liver microsomes containing the phenobarbital-induced form of cytochrome P-450, produced a typical type I binding spectrum (an absorption maximum at 390 nm; an absorption minimum at 420 nm). Studies with microsomal preparations containing cytochrome P-448, induced by 3-methylcholanthrene or beta-naphthoflavone, revealed that this hemeprotein was more susceptible to direct degradation by bis(tri-n-butyltin)oxide than was the uninduced or phenobarbital-induced forms of cytochrome P-450. The disappearance of spectrally detectable cytochrome P-450 was accompanied by an increase in cytochrome P-420. The formation of cytochrome P-420 was both time and temperature dependent, and it also occurred to a greater extent in microsomal preparations containing cytochrome P-448 than in microsomes containing the phenobarbital-induced form of cytochrome P-450. In all cases, the decreases in spectrally detectable cytochrome P-450 produced by the organotin were not accompanied by decreases in microsomal heme or cytochrome b5 content. The findings provide evidence for the direct interaction followed by conversion of cytochrome P-450 to cytochrome P-420 produced by a trialkyltin compound in vitro, and indicate that different susceptibilities to degradation exist within the various subspecies of this hemeprotein.

Studies on the mechanism of Sn-protoporphyrin suppression of hyperbilirubinemia. Inhibition of heme oxidation and bilirubin production.

The synthetic heme analogue Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, the rate-limiting enzyme in heme degradation to bile pigment, and can entirely suppress hyperbilirubinemia in neonatal animals and significantly reduce plasma bilirubin levels in a variety of circumstances in experimental animals and man. To further explore the mechanism by which this metalloporphyrin reduces bilirubin levels in vivo, we have examined its effects on bilirubin production in bile duct-cannulated rats, in which bilirubin derived from heme catabolism is known to be rapidly excreted in bile. The administration of Sn-protoporphyrin (10-50 mumol/kg body weight) was followed by prompt (within approximately 1 h) and sustained (up to at least 18 h) decreases in bilirubin output, to levels 25-30 percent below the levels of bilirubin output in control bile fistula animals. The metalloporphyrin had no effect on bile flow or the biliary output of bile acids. Infusions of heme, which is taken up primarily in hepatocytes, or of heat-damaged erythrocytes, which are taken up in reticuloendothelial cells, resulted in marked increases in bilirubin output in bile in control animals; these increases were completely prevented or substantially diminished by Sn-protoporphyrin administration. By contrast, the metalloporphyrin did not alter the high levels of bilirubin in plasma and bile that were achieved in separate experiments by the constant (16 h) infusion of unconjugated bilirubin to bile duct-cannulated rats. Thus, Sn-protoporphyrin exerts no major effects on the metabolic disposition of preformed bilirubin. Heme oxygenase activities were markedly decreased in microsomal preparations from liver, spleen, and kidneys in these experiments, to a degree comparable to the decreases we have observed in the intact rat. We also demonstrated that a substantial proportion (19-35%) of a dose of Sn-protoporphyrin is promptly excreted in bile and that the time course of biliary excretion of this compound more closely reflects plasma concentrations of the metalloporphyrin, which decline rapidly, rather than concentrations in liver, which are considerably more persistent. These results indicate that Sn-protoporphyrin substantially reduces the in vivo production of bilirubin from the degradation of endogenous as well as exogenous heme in the rat. Moreover, this inhibitory effect of the synthetic metalloporphyrin on bilirubin production occurs in both hepatocytes and reticuloendothelial cells, which are the major tissue sites for bilirubin formation. In other studies, we have established that heme oxygenase blockade by Sn-protoporphyrin leads to a marked and rapid excretion of heme into bile presumably because the synthetic metabolism to bile pigment and making it available for excretion via the biliary system in to the gut, These studies strongly suggest that Sn-protoporphyrin diminishes hyperbilirubinemia in animals and man by inhibiting the production of the bile pigment in vivo, and that its principal mode of action involves a potent and sustained competitive inhibition of heme oxygenase.

Tin-Mesoporphyrin Inhibits Heme Oxygenase Activity and Heme-Iron Absorption in the Intestine

Long-term treatment with the heme oxygenase inhibitor tin-mesoporphyrin produces an iron deficiency anemia in rats analogous to that we reported in patients with the Crigler-Najjar type I syndrome receiving prolonged treatment with the inhibitor to ameliorate severe jaundice [Pediatrics 1992; 89: 175-182]. A dose- and time-dependent inhibition of intestinal heme oxygenase is produced by tin-mesoporphyrin which is independent of iron status of the animal. Tin-mesoporphyrin inhibits the intestinal enzyme whether administered orally or parenterally. Enzyme inhibition by either route results in diminished uptake of 59Fe from radiolabelled heme in the gut. Since tin-mesoporphyrin stimulates excretion of unmetabolized heme into bile its ability to inhibit intestinal heme oxygenase and to decrease heme-iron absorption in the gut probably accounts in part for the iron deficiency produced by the agent. The availability of an orally active agent which inhibits heme oxygenase and heme-iron absorption in the intestine may prove useful for experimental and therapeutic studies in diseases of iron metabolism.