We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1–targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1–estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.

The identification of leptin as a mediator of body weight regulation provided much initial excitement for the treatment of obesity. Unfortunately, leptin monotherapy is insufficient in reversing obesity in rodents or humans. Recent findings suggest that amylin is able to restore leptin sensitivity and when used in combination with leptin enhances body weight loss in obese rodents and humans. However, as the uniqueness of this combination therapy remains unclear, we assessed whether co-administration of leptin with other weight loss-inducing hormones equally restores leptin responsiveness in diet-induced obese (DIO) mice. Accordingly, we report here the design and characterization of a series of site-specifically enhanced leptin analogs of high potency and sustained action that, when administered in combination with exendin-4 or fibroblast growth factor 21 (FGF21), restores leptin responsiveness in DIO mice after an initial body weight loss of 30%. Using either combination, body weight loss was enhanced compared with either exendin-4 or FGF21 monotherapy, and leptin alone was sufficient to maintain the reduced body weight. In contrast, leptin monotherapy proved ineffective when identical weight loss was induced by caloric restriction alone over a comparable time. Accordingly, we find that a hypothalamic counter-regulatory response to weight loss, assessed using changes in hypothalamic agouti related peptide (AgRP) levels, is triggered by caloric restriction, but blunted by treatment with exendin-4. We conclude that leptin re-sensitization requires pharmacotherapy but does not appear to be restricted to a unique signaling pathway. Our findings provide preclinical evidence that high activity, long-acting leptin analogs are additively efficacious when used in combination with other weight-lowering agents.

The scaffold protein p62 (sequestosome 1; SQSTM1) is an emerging key molecular link among the metabolic, immune, and proliferative processes of the cell. Here, we report that adipocyte-specific, but not CNS-, liver-, muscle-, or myeloid-specific p62-deficient mice are obese and exhibit a decreased metabolic rate caused by impaired nonshivering thermogenesis. Our results show that p62 regulates energy metabolism via control of mitochondrial function in brown adipose tissue (BAT). Accordingly, adipocyte-specific p62 deficiency led to impaired mitochondrial function, causing BAT to become unresponsive to β-adrenergic stimuli. Ablation of p62 leads to decreased activation of p38 targets, affecting signaling molecules that control mitochondrial function, such as ATF2, CREB, PGC1α, DIO2, NRF1, CYTC, COX2, ATP5β, and UCP1. p62 ablation in HIB1B and BAT primary cells demonstrated that p62 controls thermogenesis in a cell-autonomous manner, independently of brown adipocyte development or differentiation. Together, our data identify p62 as a novel regulator of mitochondrial function and brown fat thermogenesis.

Diabetic nephropathy (DN) is one of the main causes of end stage renal disease (ESRD) and a leading cause of diabetes mellitus related morbidity and mortality. Recently, sirtuin are reported to have emerging pathogenetic roles in cancer, muscle differentiation, heart failure, neurodegeneration, diabetes and aging. The aim of the present study was to study the role of intermittent fasting (IF) on DN and studying the expression of Sir2 and p53. At biochemical level, we found that IF causes significant improvement in blood urea nitrogen (BUN), creatinine, albumin and HDL cholesterol, parameters that are associated with the development of DN. Diabetic rats on IF also show significant improvement in onset of hypertension. Interestingly, the expression of Sir2, a NAD dependent histone deacetylase, decreases in diabetic rat kidney and this decrease is overcome by IF. Moreover, we provide evidence for involvement of mitogen activated protein kinases (MAPK) cascade in mediating the effects of IF as there is reduction in the expression of p38 which gets induced under diabetic condition. This was further accompanied by the concomitant decrease in cleavage of caspase3 and p53 expression. These findings suggest that IF significantly improves biochemical parameters associated with development of DN and changes the expression of Sir2 and p53.

Background and purpose: Curcumin has been used to treat cancer, diabetes and other pathologies. However, little is known regarding its role in altering post-translational modifications of histone H3. A recent report suggests that acute hyperglycaemia induces a global down-regulation of gene expression in human tissues and epigenetic regulation of gene expression could be a novel mechanism underlying the pathological processes of hyperglycaemia. The present study was undertaken to examine changes in histone modification by curcumin treatment which prevents development of type I diabetic nephropathy. Experimental approach: Male Sprague-Dawley rats were rendered diabetic using a single dose of streptozotocin (55 mg kg À1 , i.p.). Diabetic nephropathy was assessed by measurements of blood urea nitrogen, albumin and creatinine levels. Posttranslational modifications of histone H3, heat shock protein-27 (HSP-27) and mitogen-activated protein (MAP) kinase p38 expression were examined by western blotting. Key results: Treatment of diabetic rats with curcumin significantly decreased blood urea nitrogen and creatinine and increased albumin; variables associated with the development of diabetic nephropathy. There were also increased levels of HSP-27 and MAP kinase (p38) in diabetic kidney. However, curcumin treatment prevented this increase in HSP-27 and p38 expression. Moreover, at nuclear level curcumin prevented the decrease in dephosphorylation and increases acetylation of histone H3. Conclusions and implications: Our results suggested that protection against development of diabetic nephropathy by curcumin treatment involved changes in post-translational modifications of histone H3, expression of HSP-27 and MAP kinase p38 in diabetic kidney.

Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment.

Canonical protein phosphatase 3/calcineurin signaling is central to numerous physiological processes. Here we provide evidence that calcineurin plays a pivotal role in controlling systemic energy and body weight homeostasis. Knockdown of calcineurin in Drosophila melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure. In mice, global deficiency of catalytic subunit Ppp3cb, and tissue-specific ablation of regulatory subunit Ppp3r1 from skeletal muscle, but not adipose tissue or liver, led to protection from high-fat-diet-induced obesity and comorbid sequelæ. Ser637 hyperphosphorylation of dynamin-related protein 1 (Drp1) in skeletal muscle of calcineurin-deficient mice was associated with mitochondrial elongation into power-cable-shaped filaments and increased mitochondrial respiration, but also with attenuated exercise performance. Our data suggest that calcineurin acts as highly conserved pivot for the adaptive metabolic responses to environmental changes such as high-fat, high-sugar diets or exercise.

Celastrol, a plant-derived constituent of traditional Chinese medicine, has been proposed to offer significant potential as an antiobesity drug. However, the molecular mechanism for this activity is unknown. We show that the weight-lowering effects of celastrol are driven by decreased food consumption. Although young Lep mice respond with a decrease in food intake and body weight, adult Lep and Lep mice are unresponsive to celastrol, suggesting that functional leptin signaling in adult mice is required to elicit celastrol's catabolic actions. Protein tyrosine phosphatase 1 (PTP1B), a leptin negative-feedback regulator, has been previously reported to be one of celastrol's targets. However, we found that global PTP1B knockout (KO) and wild-type (WT) mice have comparable weight loss and hypophagia when treated with celastrol. Increased levels of uncoupling protein 1 (UCP1) in subcutaneous white and brown adipose tissue suggest celastrol-induced thermogenesis as a further mechanism. However, diet-induced obese UCP1 WT and KO mice have comparable weight loss upon celastrol treatment, and celastrol treatment has no effect on energy expenditure under ambient housing or thermoneutral conditions. Overall, our results suggest that celastrol-induced weight loss is hypophagia driven and age-dependently mediated by functional leptin signaling. Our data encourage reconsideration of therapeutic antiobesity strategies built on leptin sensitization.