Skeletal muscle and intermuscular adipose tissue gene expression profiling identifies new biomarkers with prognostic significance for insulin resistance progression and intervention response.

AIMS/HYPOTHESIS: Although insulin resistance often leads to type 2 diabetes mellitus, its early stages are often unrecognised, thus reducing the probability of successful prevention and intervention. Moreover, treatment efficacy is affected by the genetics of the individual. We used gene expression profiles from a cross-sectional study to identify potential candidate genes for the prediction of diabetes risk and intervention response. METHODS: Using a multivariate regression model, we linked gene expression profiles of human skeletal muscle and intermuscular adipose tissue (IMAT) to fasting glucose levels and glucose infusion rate. Based on the expression patterns of the top predictive genes, we characterised and compared individual gene expression with clinical classifications using k-nearest neighbour clustering. The predictive potential of the candidate genes identified was validated using muscle gene expression data from a longitudinal intervention study. RESULTS: We found that genes with a strong association with clinical measures clustered into three distinct expression patterns. Their predictive values for insulin resistance varied substantially between skeletal muscle and IMAT. Moreover, we discovered that individual gene expression-based classifications may differ from classifications based predominantly on clinical variables, indicating that participant stratification may be imprecise if only clinical variables are used for classification. Of the 15 top candidate genes, ST3GAL2, AASS, ARF1 and the transcription factor SIN3A are novel candidates for predicting a refined diabetes risk and intervention response. CONCLUSION/INTERPRETATION: Our results confirm that disease progression and successful intervention depend on individual gene expression states. We anticipate that our findings may lead to a better understanding and prediction of individual diabetes risk and may help to develop individualised intervention strategies.

GIP receptor agonism improves dyslipidemia and atherosclerosis independently of body weight loss in preclinical mouse model for cardio-metabolic disease.

BACKGROUND: Agonism at the receptor for the glucose-dependent insulinotropic polypeptide (GIPR) is a key component of the novel unimolecular GIPR:GLP-1R co-agonists, which are among the most promising drugs in clinical development for the treatment of obesity and type 2 diabetes. The therapeutic effect of chronic GIPR agonism to treat dyslipidemia and thus to reduce the cardiovascular disease risk independently of body weight loss has not been explored yet. METHODS: After 8 weeks on western diet, LDL receptor knockout (LDLR-/-) male mice were treated with daily subcutaneous injections of long-acting acylated GIP analog (acyl-GIP; 10nmol/kg body weight) for 28 days. Body weight, food intake, whole-body composition were monitored throughout the study. Fasting blood glucose and intraperitoneal glucose tolerance test (ipGTT) were determined on day 21 of the study. Circulating lipid levels, lipoprotein profiles and atherosclerotic lesion size was assessed at the end of the study. Acyl-GIP effects on fat depots were determined by histology and transcriptomics. RESULTS: Herein we found that treatment with acyl-GIP reduced dyslipidemia and atherogenesis in male LDLR-/- mice. Acyl-GIP administration resulted in smaller adipocytes within the inguinal fat depot and RNAseq analysis of the latter revealed that acyl-GIP may improve dyslipidemia by directly modulating lipid metabolism in this fat depot. CONCLUSIONS: This study identified an unanticipated efficacy of chronic GIPR agonism to improve dyslipidemia and cardiovascular disease independently of body weight loss, indicating that treatment with acyl-GIP may be a novel approach to alleviate cardiometabolic disease.

Plasma proteome profiles treatment efficacy of incretin dual agonism in diet-induced obese female and male mice.

AIMS: Unimolecular peptides targeting the receptors for glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (GLP-1/GIP co-agonist) have been shown to outperform each single peptide in the treatment of obesity and cardiometabolic disease in preclinical and clinical trials. By combining physiological treatment endpoints with plasma proteomic profiling (PPP), we aimed to identify biomarkers to advance non-invasive metabolic monitoring of compound treatment success and exploration of ulterior treatment effects on an individual basis. MATERIALS AND METHODS: We performed metabolic phenotyping along with PPP in body weight-matched male and female diet-induced obese (DIO) mice treated for 21 days with phosphate-buffered saline, single GIP and GLP-1 mono-agonists, or a GLP-1/GIP co-agonist. RESULTS: GLP-1R/GIPR co-agonism improved obesity, glucose intolerance, non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia with superior efficacy in both male and female mice compared with mono-agonist treatments. PPP revealed broader changes of plasma proteins after GLP-1/GIP co-agonist compared with mono-agonist treatments in both sexes, including established and potential novel biomarkers for systemic inflammation, NAFLD and atherosclerosis. Subtle sex-specific differences have been observed in metabolic phenotyping and PPP. CONCLUSIONS: We herein show that a recently developed unimolecular GLP-1/GIP co-agonist is more efficient in improving metabolic disease than either mono-agonist in both sexes. PPP led to the identification of a sex-independent protein panel with the potential to monitor non-invasively the treatment efficacies on metabolic function of this clinically advancing GLP-1/GIP co-agonist.

Pharmacological targeting of α3ß4 nicotinic receptors improves peripheral insulin sensitivity in mice with diet-induced obesity.

AIMS/HYPOTHESIS: Treatment with the α3ß4 nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), improves glucose tolerance in diet-induced obese (DIO) mice, but the physiological and molecular mechanisms are unknown. METHODS: DMPP (10 mg/kg body weight, s.c.) was administered either in a single injection (acute) or daily for up to 14 days (chronic) in DIO wild-type (WT) and Chrnb4 knockout (KO) mice and glucose tolerance, tissue-specific tracer-based glucose metabolism, and insulin signalling were assessed. RESULTS: In WT mice, but not in Chrnb4 KO mice, single acute treatment with DMPP induced transient hyperglycaemia, which was accompanied by high plasma adrenaline (epinephrine) levels, upregulated hepatic gluconeogenic genes, and decreased hepatic glycogen content. In contrast to these acute effects, chronic DMPP treatment in WT mice elicited improvements in glucose tolerance already evident after three consecutive days of DMPP treatment. After seven days of DMPP treatment, glucose tolerance was markedly improved, also in comparison with mice that were pair-fed to DMPP-treated mice. The glycaemic benefit of chronic DMPP was absent in Chrnb4 KO mice. Chronic DMPP increased insulin-stimulated glucose clearance into brown adipose tissue (+69%), heart (+93%), gastrocnemius muscle (+74%) and quadriceps muscle (+59%), with no effect in white adipose tissues. After chronic DMPP treatment, plasma adrenaline levels did not increase following an injection with DMPP. In glucose-stimulated skeletal muscle, we detected a decreased phosphorylation of the inhibitory Ser640 phosphorylation site on glycogen synthase and a congruent increase in glycogen accumulation following chronic DMPP treatment. CONCLUSIONS/INTERPRETATION: Our data suggest that DMPP acutely induces adrenaline release and hepatic glycogenolysis, while chronic DMPP-mediated activation of ß4-containing nAChRs improves peripheral insulin sensitivity independently of changes in body weight via mechanisms that could involve increased non-oxidative glucose disposal into skeletal muscle.

Plasma proteome profiling discovers novel proteins associated with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) affects 25% of the population and can progress to cirrhosis with limited treatment options. As the liver secretes most of the blood plasma proteins, liver disease may affect the plasma proteome. Plasma proteome profiling of 48 patients with and without cirrhosis or NAFLD revealed six statistically significantly changing proteins (ALDOB, APOM, LGALS3BP, PIGR, VTN, and AFM), two of which are already linked to liver disease. Polymeric immunoglobulin receptor (PIGR) was significantly elevated in both cohorts by 170% in NAFLD and 298% in cirrhosis and was further validated in mouse models. Furthermore, a global correlation map of clinical and proteomic data strongly associated DPP4, ANPEP, TGFBI, PIGR, and APOE with NAFLD and cirrhosis. The prominent diabetic drug target DPP4 is an aminopeptidase like ANPEP, ENPEP, and LAP3, all of which are up-regulated in the human or mouse data. Furthermore, ANPEP and TGFBI have potential roles in extracellular matrix remodeling in fibrosis. Thus, plasma proteome profiling can identify potential biomarkers and drug targets in liver disease.

Intermuscular adipose tissue directly modulates skeletal muscle insulin sensitivity in humans.

Intermuscular adipose tissue (IMAT) is negatively related to insulin sensitivity, but a causal role of IMAT in the development of insulin resistance is unknown. IMAT was sampled in humans to test for the ability to induce insulin resistance in vitro and characterize gene expression to uncover how IMAT may promote skeletal muscle insulin resistance. Human primary muscle cells were incubated with conditioned media from IMAT, visceral (VAT), or subcutaneous adipose tissue (SAT) to evaluate changes in insulin sensitivity. RNAseq analysis was performed on IMAT with gene expression compared with skeletal muscle and SAT, and relationships to insulin sensitivity were determined in men and women spanning a wide range of insulin sensitivity measured by hyperinsulinemic-euglycemic clamp. Conditioned media from IMAT and VAT decreased insulin sensitivity similarly compared with SAT. Multidimensional scaling analysis revealed distinct gene expression patterns in IMAT compared with SAT and muscle. Pathway analysis revealed that IMAT expression of genes in insulin signaling, oxidative phosphorylation, and peroxisomal metabolism related positively to donor insulin sensitivity, whereas expression of macrophage markers, inflammatory cytokines, and secreted extracellular matrix proteins were negatively related to insulin sensitivity. Perilipin 5 gene expression suggested greater IMAT lipolysis in insulin-resistant individuals. Combined, these data show that factors secreted from IMAT modulate muscle insulin sensitivity, possibly via secretion of inflammatory cytokines and extracellular matrix proteins, and by increasing local FFA concentration in humans. These data suggest IMAT may be an important regulator of skeletal muscle insulin sensitivity and could be a novel therapeutic target for skeletal muscle insulin resistance.

Glucagon Regulation of Energy Expenditure.

Glucagon's ability to increase energy expenditure has been known for more than 60 years, yet the mechanisms underlining glucagon's thermogenic effect still remain largely elusive. Over the last years, significant efforts were directed to unravel the physiological and cellular underpinnings of how glucagon regulates energy expenditure. In this review, we summarize the current knowledge on how glucagon regulates systems metabolism with a special emphasis on its acute and chronic thermogenic effects.

Circulating HDL levels control hypothalamic astrogliosis via apoA-I.

Meta-inflammation of hypothalamic areas governing energy homeostasis has recently emerged as a process of potential pathophysiological relevance for the development of obesity and its metabolic sequelae. The current model suggests that diet-induced neuronal injury triggers microgliosis and astrocytosis, conditions which ultimately may induce functional impairment of hypothalamic circuits governing feeding behavior, systemic metabolism, and body weight. Epidemiological data indicate that low circulating HDL levels, besides conveying cardiovascular risk, also correlate strongly with obesity. We simulated that condition by using a genetic loss of function mouse model (apoA-I-/-) with markedly reduced HDL levels to investigate whether HDL may directly modulate hypothalamic inflammation. Astrogliosis was significantly enhanced in the hypothalami of apoA-I-/- compared with apoA-I+/+ mice and was associated with compromised mitochondrial function. apoA-I-/- mice exhibited key components of metabolic disease, like increased fat mass, fasting glucose levels, hepatic triglyceride content, and hepatic glucose output compared with apoA-I+/+ controls. Administration of reconstituted HDL (CSL-111) normalized hypothalamic inflammation and mitochondrial function markers in apoA-I-/- mice. Treatment of primary astrocytes with apoA-I resulted in enhanced mitochondrial activity, implying that circulating HDL levels are likely important for astrocyte function. HDL-based therapies may consequently avert reactive gliosis in hypothalamic astrocytes by improving mitochondrial bioenergetics and thereby offering potential treatment and prevention for obesity and metabolic disease.

Pharmacological Targeting of Endoplasmic Reticulum Stress in Pancreatic Beta Cells.

Diabetes is a disease with pandemic dimensions and no pharmacological treatment prevents disease progression. Dedifferentiation has been proposed to be a driver of beta-cell dysfunction in both type 1 and type 2 diabetes. Regenerative therapies aim to re-establish function in dysfunctional or dedifferentiated beta cells and restore the defective insulin secretion. Unsustainable levels of insulin production, with increased demand at disease onset, strain the beta-cell secretory machinery, leading to endoplasmic reticulum (ER) stress. Unresolved chronic ER stress is a major contributor to beta-cell loss of function and identity. Restoring ER homeostasis, enhancing ER-associated degradation of misfolded protein, and boosting chaperoning activity, are emerging therapeutic approaches for diabetes treatment.

A point mutation in the Pdia6 gene results in loss of pancreatic ß-cell identity causing overt diabetes.

OBJECTIVE: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in ß-cell dysfunction and diabetes. However, very little is known about the function of this protein in ß-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on ß-cell development and function. METHODS: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the ß-cell development at embryonic day (E)18.5 and ß-cell identity as well as function at postnatal stages. RESULTS: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing ß-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of ß-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of ß-cell identity. CONCLUSIONS: The results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic ß-cell function and identity, suggesting a critical role of PDIA6 specifically for ß-cells.

Targeted pharmacological therapy restores ß-cell function for diabetes remission.

Dedifferentiation of insulin-secreting ß cells in the islets of Langerhans has been proposed to be a major mechanism of ß-cell dysfunction. Whether dedifferentiated ß cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study ß-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with ß-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in ß cells and restores maturation and function for diabetes remission. Additional ß-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases ß-cell survival and regeneration. GLP-1-oestrogen also protects human ß cells against cytokine-induced dysfunction. This study not only describes mechanisms of ß-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated ß cells for diabetes remission.

The scaffold protein p62 regulates adaptive thermogenesis through ATF2 nuclear target activation.

During ß-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62-/- and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.

Preadipocytes of obese humans display gender-specific bioenergetic responses to glucose and insulin.

BACKGROUND/OBJECTIVES: Although the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age- and BMI-matched) donors must exist. SUBJECTS/METHODS: Using Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined. RESULTS: Dissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity. CONCLUSIONS: Collectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose.

Publisher Correction: Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes.

In the original PDF version of this article, affiliation 1, 'Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany', was incorrectly given as 'Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany '. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.

Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes.

Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3ß4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3ß4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.

Monomeric GLP-1/GIP/glucagon triagonism corrects obesity, hepatosteatosis, and dyslipidemia in female mice.

OBJECTIVE: Obesity is a major health threat that affects men and women equally. Despite this fact, weight-loss potential of pharmacotherapies is typically first evaluated in male mouse models of diet-induced obesity (DIO). To address this disparity we herein determined whether a monomeric peptide with agonism at the receptors for glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon is equally efficient in correcting DIO, dyslipidemia, and glucose metabolism in DIO female mice as it has been previously established for DIO male mice. METHODS: Female C57BL/6J mice and a cohort of fatmass-matched C57BL/6J male mice were treated for 27 days via subcutaneous injections with either the GLP-1/GIP/glucagon triagonist or PBS. A second cohort of C57BL/6J male mice was included to match the females in the duration of the high-fat, high-sugar diet (HFD) exposure. RESULTS: Our results show that GLP-1/GIP/glucagon triple agonism inhibits food intake and decreases body weight and body fat mass with comparable potency in male and female mice that have been matched for body fat mass. Treatment improved dyslipidemia in both sexes and reversed diet-induced steatohepatitis to a larger extent in female mice compared to male mice. CONCLUSIONS: We herein show that a recently developed unimolecular peptide triagonist is equally efficient in both sexes, suggesting that this polypharmaceutical strategy might be a relevant alternative to bariatric surgery for the treatment of obesity and related metabolic disorders.

Activated macrophages control human adipocyte mitochondrial bioenergetics via secreted factors.

OBJECTIVE: Obesity-associated WAT inflammation is characterized by the accumulation and local activation of macrophages (MΦs), and recent data from mouse studies suggest that macrophages are modifiers of adipocyte energy metabolism and mitochondrial function. As mitochondrial dysfunction has been associated with obesity and the metabolic syndrome in humans, herein we aimed to delineate how human macrophages may affect energy metabolism of white adipocytes. METHODS: Human adipose tissue gene expression analysis for markers of macrophage activation and tissue inflammation (CD11c, CD40, CD163, CD206, CD80, MCP1, TNFα) in relationship to mitochondrial complex I (NDUFB8) and complex III (UQCRC2) was performed on subcutaneous WAT of 24 women (BMI 20-61 kg/m2). Guided by these results, the impact of secreted factors of LPS/IFNγ- and IL10/TGFß-activated human macrophages (THP1, primary blood-derived) on mitochondrial function in human subcutaneous white adipocytes (SGBS, primary) was determined by extracellular flux analysis (Seahorse technology) and gene/protein expression. RESULTS: Stepwise regression analysis of human WAT gene expression data revealed that a linear combination of CD40 and CD163 was the strongest predictor for mitochondrial complex I (NDUFB8) and complex III (UQCRC2) levels, independent of BMI. IL10/TGFß-activated MΦs displayed high CD163 and low CD40 expression and secreted factors that decreased UQCRC2 gene/protein expression and ATP-linked respiration in human white adipocytes. In contrast, LPS/IFNγ-activated MΦs showed high CD40 and low CD163 expression and secreted factors that enhanced adipocyte mitochondrial activity resulting in a total difference of 37% in ATP-linked respiration of white adipocytes (p = 0.0024) when comparing the effect of LPS/IFNγ- vs IL10/TGFß-activated MΦs. CONCLUSION: Our data demonstrate that macrophages modulate human adipocyte energy metabolism via an activation-dependent paracrine mechanism.