Impaired mitochondrial fatty acid oxidation and insulin resistance in aging: novel protective role of glutathione.

Aging is associated with impaired fasted oxidation of nonesterified fatty acids (NEFA) suggesting a mitochondrial defect. Aging is also associated with deficiency of glutathione (GSH), an important mitochondrial antioxidant, and with insulin resistance. This study tested whether GSH deficiency in aging contributes to impaired mitochondrial NEFA oxidation and insulin resistance, and whether GSH restoration reverses these defects. Three studies were conducted: (i) in 82-week-old C57BL/6 mice, the effect of naturally occurring GSH deficiency and its restoration on mitochondrial (13) C1 -palmitate oxidation and glucose metabolism was compared with 22-week-old C57BL/6 mice; (ii) in 20-week C57BL/6 mice, the effect of GSH depletion on mitochondrial oxidation of (13) C1 -palmitate and glucose metabolism was studied; (iii) the effect of GSH deficiency and its restoration on fasted NEFA oxidation and insulin resistance was studied in GSH-deficient elderly humans, and compared with GSH-replete young humans. Chronic GSH deficiency in old mice and elderly humans was associated with decreased fasted mitochondrial NEFA oxidation and insulin resistance, and these defects were reversed with GSH restoration. Acute depletion of GSH in young mice resulted in lower mitochondrial NEFA oxidation, but did not alter glucose metabolism. These data suggest that GSH is a novel regulator of mitochondrial NEFA oxidation and insulin resistance in aging. Chronic GSH deficiency promotes impaired NEFA oxidation and insulin resistance, and GSH restoration reverses these defects. Supplementing diets of elderly humans with cysteine and glycine to correct GSH deficiency could provide significant metabolic benefits.

Berardinelli-seip congenital lipodystrophy 2/seipin is a cell-autonomous regulator of lipolysis essential for adipocyte differentiation.

Mutations in BSCL2 underlie human congenital generalized lipodystrophy. We inactivated Bscl2 in mice to examine the mechanisms whereby absence of Bscl2 leads to adipose tissue loss and metabolic disorders. Bscl2(-/-) mice develop severe lipodystrophy of white adipose tissue (WAT), dyslipidemia, insulin resistance, and hepatic steatosis. In vitro differentiation of both Bscl2(-/-) murine embryonic fibroblasts (MEFs) and stromal vascular cells (SVCs) reveals normal early-phase adipocyte differentiation but a striking failure in terminal differentiation due to unbridled cyclic AMP (cAMP)-dependent protein kinase A (PKA)-activated lipolysis, which leads to loss of lipid droplets and silencing of the expression of adipose tissue-specific transcription factors. Importantly, such defects in differentiation can be largely rescued by inhibitors of lipolysis but not by a gamma peroxisome proliferator-activated receptor (PPARγ) agonist. The residual epididymal WAT (EWAT) in Bscl2(-/-) mice displays enhanced lipolysis. It also assumes a "brown-like" phenotype with marked upregulation of UCP1 and other brown adipose tissue-specific markers. Together with decreased Pref1 but increased C/EBPß levels, these changes highlight a possible increase in cAMP signaling that impairs terminal adipocyte differentiation in the EWAT of Bscl2(-/-) mice. Our study underscores the fundamental role of regulated cAMP/PKA-mediated lipolysis in adipose differentiation and identifies Bscl2 as a novel cell-autonomous determinant of activated lipolysis essential for terminal adipocyte differentiation.

Glutathione synthesis is diminished in patients with uncontrolled diabetes and restored by dietary supplementation with cysteine and glycine.

OBJECTIVE: Sustained hyperglycemia is associated with low cellular levels of the antioxidant glutathione (GSH), which leads to tissue damage attributed to oxidative stress. We tested the hypothesis that diminished GSH in adult patients with uncontrolled type 2 diabetes is attributed to decreased synthesis and measured the effect of dietary supplementation with its precursors cysteine and glycine on GSH synthesis rate and oxidative stress. RESEARCH DESIGN AND METHODS: We infused 12 diabetic patients and 12 nondiabetic control subjects with [²H2]-glycine to measure GSH synthesis. We also measured intracellular GSH concentrations, reactive oxygen metabolites, and lipid peroxides. Diabetic patients were restudied after 2 weeks of dietary supplementation with the GSH precursors cysteine and glycine. RESULTS: Compared with control subjects, diabetic subjects had significantly higher fasting glucose (5.0 ± 0.1 vs. 10.7 ± 0.5 mmol/l; P < 0.001), lower erythrocyte concentrations of glycine (514.7 ± 33.1 vs. 403.2 ± 18.2 µmol/l; P < 0.01), and cysteine (25.2 ± 1.5 vs. 17.8 ± 1.5 µmol/l; P < 0.01); lower concentrations of GSH (6.75 ± 0.47 vs. 1.65 ± 0.16 µmol/g Hb; P < 0.001); diminished fractional (79.21 ± 5.75 vs. 44.86 ± 2.87%/day; P < 0.001) and absolute (5.26 ± 0.61 vs. 0.74 ± 0.10 µmol/g Hb/day; P < 0.001) GSH synthesis rates; and higher reactive oxygen metabolites (286 ± 10 vs. 403 ± 11 Carratelli units [UCarr]; P < 0.001) and lipid peroxides (2.6 ± 0.4 vs. 10.8 ± 1.2 pg/ml; P < 0.001). Following dietary supplementation in diabetic subjects, GSH synthesis and concentrations increased significantly and plasma oxidative stress and lipid peroxides decreased significantly. CONCLUSIONS: Patients with uncontrolled type 2 diabetes have severely deficient synthesis of glutathione attributed to limited precursor availability. Dietary supplementation with GSH precursor amino acids can restore GSH synthesis and lower oxidative stress and oxidant damage in the face of persistent hyperglycemia.

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic-acid methyl ester has potent anti-diabetic effects in diet-induced diabetic mice and Lepr(db/db) mice.

The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic-acid (CDDO) and its methyl ester (CDDO-Me) are undergoing clinical trials in cancer and leukemia therapy. Here we report that CDDO-Me ameliorates diabetes in high fat diet-fed type 2 diabetic mice and in Lepr(db/db) mice. CDDO-Me reduces proinflammatory cytokine expression in these animals. Oral CDDO-Me administration reduces total body fat, plasma triglyceride, and free fatty acid levels. It also improves glucose tolerance and insulin tolerance tests. Its potent glucose-lowering activity results from enhanced insulin action. Hyperinsulinemic-euglycemic clamp reveals an increased glucose infusion rate required to maintain euglycemia and showed a significant increase in muscle-specific insulin-stimulated glucose uptake (71% soleus, 58% gastrocnemius) and peripheral glucose clearance as documented by a 48% increase in glucose disposal rate. CDDO-Me activates AMP-activated protein kinase (AMPK) and via LKB1 activation in muscle and liver in vivo. Treatment of isolated hepatocytes with CDDO-Me directly stimulates AMPK activity and LKB1 phosphorylation and decreases acetyl-coA carboxylase activity; it also down-regulates lipogenic gene expression, suppresses gluconeogenesis, and increases glucose uptake. Inhibition of AMPK phosphorylation using compound C and lentiviral-mediated knockdown of AMPK completely blocks the CDDO-Me-induced effect on hepatocytes as well as C(2)C(12) cells. We conclude that the triterpenoid CDDO-Me has potent anti-diabetic action in diabetic mouse models that is mediated at least in part through AMPK activation. The in vivo anti-diabetogenic effects occur at a dose substantially lower than that used for anti-leukemia therapy. We suggest that CDDO-Me holds promise as a potential anti-diabetic agent.

Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass.

Overexpression of hepatocyte growth factor (HGF) in the beta-cell of transgenic mice enhances beta-cell proliferation, survival, and function. In the current studies, we have used conditional ablation of the c-met gene to uncover the physiological role of HGF in beta-cell growth and function. Mice in which c-met is inactivated in the beta-cell (MetCKO mice) display normal body weight, blood glucose, and plasma insulin compared with control littermates. In contrast, MetCKO mice displayed significantly diminished glucose tolerance and reduced plasma insulin after a glucose challenge in vivo. This impaired glucose tolerance in MetCKO mice was not caused by insulin resistance because sensitivity to exogenous insulin was similar in both groups. Importantly, in vitro glucose-stimulated insulin secretion in MetCKO islets was decreased by approximately 50% at high glucose concentrations compared with control islets. Furthermore, whereas insulin and glucokinase expression in MetCKO islets were normal, GLUT-2 expression was decreased by approximately 50%. These changes in beta-cell function in MetCKO mice were not accompanied by changes in total beta-cell mass, islet morphology, islet cell composition, and beta-cell proliferation. Interestingly, however, MetCKO mice display an increased number of small islets, mainly single and doublet beta-cells. We conclude that HGF/c-met signaling in the beta-cell is not essential for beta-cell growth, but it is essential for normal glucose-dependent insulin secretion.

Prevention of acute ischemic renal failure by targeted delivery of growth factors to the proximal tubule in transgenic mice: the efficacy of parathyroid hormone-related protein and hepatocyte growth factor.

Treatment of acute renal failure (ARF) would be enhanced by identification of factors that accelerate renal recovery from injury. Parathyroid hormone-related protein (PTHrP) and hepatocyte growth factor (HGF) have been shown to stimulate proliferation in proximal nephron-derived cells. For studying the pathophysiologic roles and therapeutic potential of these two factors in ARF, transgenic mice overexpressing PTHrP or HGF in the proximal tubule under the direction of the gamma-glutamyl transpeptidase-I promoter were developed. These mice display (1) abundant expression of the respective transgenes in the kidney; (2) similar PTH type I receptor and HGF receptor (c-met) expression levels in the proximal tubule compared with control littermates; and (3) normal renal morphology, function, and tubule cell proliferation under basal conditions. However, in contrast to control mice, when acute ischemic renal injury was induced, renal function rapidly and dramatically recovered in HGF-overexpressing mice. In addition, 48 h after ischemia, HGF-overexpressing transgenic mice displayed a fourfold increase in tubule cell proliferation and a threefold decrease in apoptotic tubule cell death compared with control mice. In contrast, PTHrP-overexpressing mice responded to either ischemic or folic acid-induced renal damage similarly to control mice. These studies demonstrate that overexpression of PTHrP in the proximal nephron of mice does not seem to provide protection against acute renal injury. In marked contrast, HGF overexpression results in dramatic protection from ischemia-induced ARF, without inducing any apparent alteration in the physiology of the kidney under normal conditions. These studies suggest that HGF, when targeted specifically to the proximal tubule, may have therapeutic potential in providing protection against ischemia-induced renal failure.

Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death.

Hepatocyte growth factor (HGF) increases beta cell proliferation and function in rat insulin promoter (RIP)-targeted transgenic mice. RIP-HGF mouse islets also function superiorly to normal islets in a transplant setting. Here, we aimed to determine whether viral gene transfer of the HGF gene into mouse islets ex vivo could enhance the performance of normal islets in a streptozotocin-diabetic severe combined immunodeficient mouse marginal islet mass model in which 300 uninfected or adenovirus (Adv) LacZ-transduced islet equivalents were insufficient to correct hyperglycemia. In dramatic contrast, 300 AdvHGF-transduced islet equivalents promptly (day 1) and significantly (p < 0.01) decreased random non-fasting blood glucose levels, from 351 +/- 20 mg/dl to an average of 191 +/- 7 mg/dl over 8 weeks. At day 1 post-transplant, beta cell death was significantly (p < 0.05) decreased, and the total insulin content was significantly (p < 0.05) increased in AdvHGF-transduced islets containing grafts. This anti-beta cell death action of HGF was independently confirmed in RIP-HGF mice and in INS-1 cells, both treated with streptozotocin. Activation of the phosphatidylinositol 3-kinase/Akt intracellular-signaling pathway appeared to be involved in this beta cell protective effect of HGF in vitro. In summary, adenoviral delivery of HGF to murine islets ex vivo improves islet transplant survival and blood glucose control in a subcapsular renal graft model in immuno-incompetent diabetic mice.