GDF15 neutralization restores muscle function and physical performance in a mouse model of cancer cachexia.

Cancer cachexia is a disorder characterized by involuntary weight loss and impaired physical performance. Decline in physical performance of patients with cachexia is associated with poor quality of life, and currently there are no effective pharmacological interventions that restore physical performance. Here we examine the effect of GDF15 neutralization in a mouse model of cancer-induced cachexia (TOV21G) that manifests weight loss and muscle function impairments. With comprehensive assessments, our results demonstrate that cachectic mice treated with the anti-GDF15 antibody mAB2 exhibit body weight gain with near-complete restoration of muscle mass and markedly improved muscle function and physical performance. Mechanistically, the improvements induced by GDF15 neutralization are primarily attributed to increased caloric intake, while altered gene expression in cachectic muscles is restored in caloric-intake-dependent and -independent manners. The findings indicate potential of GDF15 neutralization as an effective therapy to enhance physical performance of patients with cachexia.

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.

OBJECTIVE: Diabetes is characterized by pancreatic ß-cell dedifferentiation. Dedifferentiating ß cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the ß-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear. METHODS: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to ß-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios. RESULTS: The expression of Cyb5r3 is decreased in FoxO1-deficient ß cells. Mice with ß-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient ß cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient ß cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability. CONCLUSIONS: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate ß-cell failure.

Pair Feeding, but Not Insulin, Phloridzin, or Rosiglitazone Treatment, Curtails Markers of ß-Cell Dedifferentiation in db/db Mice.

ß-Cell failure is a hallmark of type 2 diabetes. Among several cellular biological mechanisms of cellular dysfunction, we and others have recently proposed that dedifferentiation of ß-cells can explain the slowly progressive onset and partial reversibility of ß-cell failure. Accordingly, we provided evidence of such processes in humans and experimental animal models of insulin-resistant diabetes. In this study, we asked whether ß-cell dedifferentiation can be prevented with diet or pharmacological treatment of diabetes. db/db mice, a widely used model of insulin-resistant diabetes and obesity, were either pair fed or treated with the Sglt inhibitor phloridzin, the insulin-sensitizer rosiglitazone, or insulin. All treatments were equally efficacious in reducing plasma glucose levels. Pair feeding and phloridzin also resulted in significant weight loss. However, pair feeding among the four treatments resulted in a reduction of ß-cell dedifferentiation, as assessed by Foxo1 and Aldh1a3 immunohistochemistry. The effect of diet to partly restore ß-cell function is consistent with data in human diabetes and provides another potential mechanism by which lifestyle changes act as an effective intervention against diabetes progression.

Aldehyde dehydrogenase 1a3 defines a subset of failing pancreatic ß cells in diabetic mice.

Insulin-producing ß cells become dedifferentiated during diabetes progression. An impaired ability to select substrates for oxidative phosphorylation, or metabolic inflexibility, initiates progression from ß-cell dysfunction to ß-cell dedifferentiation. The identification of pathways involved in dedifferentiation may provide clues to its reversal. Here we isolate and functionally characterize failing ß cells from various experimental models of diabetes and report a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (ALDH(+)) as ß cells become dedifferentiated. Flow-sorted ALDH(+) islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrates that ALDH(+) cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of ß-cell failure.

Pathological Type-2 Immune Response, Enhanced Tumor Growth, and Glucose Intolerance in Retnlß (RELMß) Null Mice: A Model of Intestinal Immune System Dysfunction in Disease Susceptibility.

Resistin, and its closely related homologs, the resistin-like molecules (RELMs) have been implicated in metabolic dysregulation, inflammation, and cancer. Specifically, RELMß, expressed predominantly in the goblet cells in the colon, is released both apically and basolaterally, and is hence found in both the intestinal lumen in the mucosal layer as well as in the circulation. RELMß has been linked to both the pathogenesis of colon cancer and type 2 diabetes. RELMß plays a complex role in immune system regulation, and the impact of loss of function of RELMß on colon cancer and metabolic regulation has not been fully elucidated. We therefore tested whether Retnlß (mouse ortholog of human RETNLß) null mice have an enhanced or reduced susceptibility for colon cancer as well as metabolic dysfunction. We found that the lack of RELMß leads to increased colonic expression of T helper cell type-2 cytokines and IL-17, associated with a reduced ability to maintain intestinal homeostasis. This defect leads to an enhanced susceptibility to the development of inflammation, colorectal cancer, and glucose intolerance. In conclusion, the phenotype of the Retnlß null mice unravels new aspects of inflammation-mediated diseases and strengthens the notion that a proper intestinal barrier function is essential to sustain a healthy phenotype.

FoxO1 Deacetylation Decreases Fatty Acid Oxidation in ß-Cells and Sustains Insulin Secretion in Diabetes.

Pancreatic ß-cell dysfunction contributes to onset and progression of type 2 diabetes. In this state ß-cells become metabolically inflexible, losing the ability to select between carbohydrates and lipids as substrates for mitochondrial oxidation. These changes lead to ß-cell dedifferentiation. We have proposed that FoxO proteins are activated through deacetylation-dependent nuclear translocation to forestall the progression of these abnormalities. However, how deacetylated FoxO exert their actions remains unclear. To address this question, we analyzed islet function in mice homozygous for knock-in alleles encoding deacetylated FoxO1 (6KR). Islets expressing 6KR mutant FoxO1 have enhanced insulin secretion in vivo and ex vivo and decreased fatty acid oxidation ex vivo Remarkably, the gene expression signature associated with FoxO1 deacetylation differs from wild type by only ∼2% of the >4000 genes regulated in response to re-feeding. But this narrow swath includes key genes required for ß-cell identity, lipid metabolism, and mitochondrial fatty acid and solute transport. The data support the notion that deacetylated FoxO1 protects ß-cell function by limiting mitochondrial lipid utilization and raise the possibility that inhibition of fatty acid oxidation in ß-cells is beneficial to diabetes treatment.

Altered Plasma Profile of Antioxidant Proteins as an Early Correlate of Pancreatic ß Cell Dysfunction.

Insulin resistance and ß cell dysfunction contribute to the pathogenesis of type 2 diabetes. Unlike insulin resistance, ß cell dysfunction remains difficult to predict and monitor, because of the inaccessibility of the endocrine pancreas, the integrated relationship with insulin sensitivity, and the paracrine effects of incretins. The goal of our study was to survey the plasma response to a metabolic challenge in order to identify factors predictive of ß cell dysfunction. To this end, we combined (i) the power of unbiased iTRAQ (isobaric tag for relative and absolute quantification) mass spectrometry with (ii) direct sampling of the portal vein following an intravenous glucose/arginine challenge (IVGATT) in (iii) mice with a genetic ß cell defect. By so doing, we excluded the effects of peripheral insulin sensitivity as well as those of incretins on ß cells, and focused on the first phase of insulin secretion to capture the early pathophysiology of ß cell dysfunction. We compared plasma protein profiles with ex vivo islet secretome and transcriptome analyses. We detected changes to 418 plasma proteins in vivo, and detected changes to 262 proteins ex vivo The impairment of insulin secretion was associated with greater overall changes in the plasma response to IVGATT, possibly reflecting metabolic instability. Reduced levels of proteins regulating redox state and neuronal stress markers, as well as increased levels of coagulation factors, antedated the loss of insulin secretion in diabetic mice. These results suggest that a reduced complement of antioxidants in response to a mixed secretagogue challenge is an early correlate of future ß cell failure.

Evidence of ß-Cell Dedifferentiation in Human Type 2 Diabetes.

CONTEXT: Diabetes is associated with a deficit of insulin-producing ß-cells. Animal studies show that ß-cells become dedifferentiated in diabetes, reverting to a progenitor-like stage, and partly converting to other endocrine cell types. OBJECTIVE: To determine whether similar processes occur in human type 2 diabetes, we surveyed pancreatic islets from 15 diabetic and 15 nondiabetic organ donors. DESIGN: We scored dedifferentiation using markers of endocrine lineage, ß-cell-specific transcription factors, and a newly identified endocrine progenitor cell marker, aldehyde dehydrogenase 1A3. RESULTS: By these criteria, dedifferentiated cells accounted for 31.9% of ß-cells in type 2 diabetics vs 8.7% in controls, and for 16.8% vs 6.5% of all endocrine cells (P < .001). The number of aldehyde dehydrogenase 1A3-positive/hormone-negative cells was 3-fold higher in diabetics compared with controls. Moreover, ß-cell-specific transcription factors were ectopically found in glucagon- and somatostatin-producing cells of diabetic subjects. CONCLUSIONS: The data support the view that pancreatic ß-cells become dedifferentiated and convert to α- and δ-"like" cells in human type 2 diabetes. The findings should prompt a reassessment of goals in the prevention and treatment of ß-cell dysfunction.

Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple FoxO-deficient mice.

Pancreatic ß cell failure in type 2 diabetes is associated with functional abnormalities of insulin secretion and deficits of ß cell mass. It's unclear how one begets the other. We have shown that loss of ß cell mass can be ascribed to impaired FoxO1 function in different models of diabetes. Here we show that ablation of the three FoxO genes (1, 3a, and 4) in mature ß cells results in early-onset, maturity-onset diabetes of the young (MODY)-like diabetes, with abnormalities of the MODY networks Hnf4α, Hnf1α, and Pdx1. FoxO-deficient ß cells are metabolically inflexible, i.e., they preferentially utilize lipids rather than carbohydrates as an energy source. This results in impaired ATP generation and reduced Ca(2+)-dependent insulin secretion. The present findings demonstrate a secretory defect caused by impaired FoxO activity that antedates dedifferentiation. We propose that defects in both pancreatic ß cell function and mass arise through FoxO-dependent mechanisms during diabetes progression.

Blunted refeeding response and increased locomotor activity in mice lacking FoxO1 in synapsin-Cre-expressing neurons.

Successful development of antiobesity agents requires detailed knowledge of neural pathways controlling body weight, eating behavior, and peripheral metabolism. Genetic ablation of FoxO1 in selected hypothalamic neurons decreases food intake, increases energy expenditure, and improves glucose homeostasis, highlighting the role of this gene in insulin and leptin signaling. However, little is known about potential effects of FoxO1 in other neurons. To address this question, we executed a broad-based neuronal ablation of FoxO1 using Synapsin promoter-driven Cre to delete floxed Foxo1 alleles. Lineage-tracing experiments showed that NPY/AgRP and POMC neurons were minimally affected by the knockout. Nonetheless, Syn-Cre-Foxo1 knockouts demonstrated a catabolic energy homeostatic phenotype with a blunted refeeding response, increased sensitivity to leptin and amino acid signaling, and increased locomotor activity, likely attributable to increased melanocortinergic tone. We confirmed these data in mice lacking the three Foxo genes. The effects on locomotor activity could be reversed by direct delivery of constitutively active FoxO1 to the mediobasal hypothalamus, but not to the suprachiasmatic nucleus. The data reveal that the integrative function of FoxO1 extends beyond the arcuate nucleus, suggesting that central nervous system inhibition of FoxO1 function can be leveraged to promote hormone sensitivity and prevent a positive energy balance.

Increased atherosclerosis and endothelial dysfunction in mice bearing constitutively deacetylated alleles of Foxo1 gene.

Complications of atherosclerosis are the leading cause of death of patients with type 2 (insulin-resistant) diabetes. Understanding the mechanisms by which insulin resistance and hyperglycemia contribute to atherogenesis in key target tissues (liver, vessel wall, hematopoietic cells) can assist in the design of therapeutic approaches. We have shown that hyperglycemia induces FoxO1 deacetylation and that targeted knock-in of alleles encoding constitutively deacetylated FoxO1 in mice (Foxo1(KR/KR)) improves hepatic lipid metabolism and decreases macrophage inflammation, setting the stage for a potential anti-atherogenic effect of this mutation. Surprisingly, we report here that when Foxo1(KR/KR) mice are intercrossed with low density lipoprotein receptor knock-out mice (Ldlr(-/-)), they develop larger aortic root atherosclerotic lesions than Ldlr(-/-) controls despite lower plasma cholesterol and triglyceride levels. The phenotype is unaffected by transplanting bone marrow from Ldlr(-/-) mice into Foxo1(KR/KR) mice, indicating that it is independent of hematopoietic cells and suggesting that the primary lesion in Foxo1(KR/KR) mice occurs in the vessel wall. Experiments in isolated endothelial cells from Foxo1(KR/KR) mice indicate that deacetylation favors FoxO1 nuclear accumulation and exerts target gene-specific effects, resulting in higher Icam1 and Tnfα expression and increased monocyte adhesion. The data indicate that FoxO1 deacetylation can promote vascular endothelial changes conducive to atherosclerotic plaque formation.

Dissociation of the glucose and lipid regulatory functions of FoxO1 by targeted knockin of acetylation-defective alleles in mice.

FoxO1 integrates multiple metabolic pathways. Nutrient levels modulate FoxO1 acetylation, but the functional consequences of this posttranslational modification are unclear. To answer this question, we generated mice bearing alleles that encode constitutively acetylated and acetylation-defective FoxO1 proteins. Homozygosity for an allele mimicking constitutive acetylation (Foxo1(KQ/KQ)) results in embryonic lethality due to cardiac and angiogenesis defects. In contrast, mice homozygous for a constitutively deacetylated Foxo1 allele (Foxo1(KR/KR)) display a unique metabolic phenotype of impaired insulin action on hepatic glucose metabolism but decreased plasma lipid levels and low respiratory quotient that are consistent with a state of preferential lipid usage. Moreover, Foxo1(KR/KR) mice show a dissociation between weight gain and insulin resistance in predisposing conditions (high fat diet, diabetes, and insulin receptor mutations), possibly due to decreased cytokine production in adipose tissue. Thus, acetylation inactivates FoxO1 during nutrient excess whereas deacetylation selectively potentiates FoxO1 activity, protecting against excessive catabolism during nutrient deprivation.

Proatherogenic abnormalities of lipid metabolism in SirT1 transgenic mice are mediated through Creb deacetylation.

Dyslipidemia and atherosclerosis are associated with reduced insulin sensitivity and diabetes, but the mechanism is unclear. Gain of function of the gene encoding deacetylase SirT1 improves insulin sensitivity and could be expected to protect against lipid abnormalities. Surprisingly, when transgenic mice overexpressing SirT1 (SirBACO) are placed on atherogenic diet, they maintain better glucose homeostasis, but develop worse lipid profiles and larger atherosclerotic lesions than controls. We show that transcription factor cAMP response element binding protein (Creb) is deacetylated in SirBACO mice. We identify Lys136 is a substrate for SirT1-dependent deacetylation that affects Creb activity by preventing its cAMP-dependent phosphorylation, leading to reduced expression of glucogenic genes and promoting hepatic lipid accumulation and secretion. Expression of constitutively acetylated Creb (K136Q) in SirBACO mice mimics Creb activation and abolishes the dyslipidemic and insulin-sensitizing effects of SirT1 gain of function. We propose that SirT1-dependent Creb deacetylation regulates the balance between glucose and lipid metabolism, integrating fasting signals.