Objective: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step.

Methods: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (n_total = 145).

Results: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations.

Conclusions: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.

Objective: Mutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo.

Methods/results: Here we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling.

Conclusions: Our findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients.

Objective: Surplus dietary fat cannot be converted into other macronutrient forms or excreted, so has to be stored or oxidized. Healthy mammals store excess energy in the form of triacylgycerol (TAG) in lipid droplets within adipocytes rather than oxidizing it, and thus ultimately gain weight. The ‘overflow hypothesis’ posits that the capacity to increase the size and number of adipocytes is finite and that when this limit is exceeded, fat accumulates in ectopic sites and leads to metabolic disease.

Methods: Here we studied the energetic and biochemical consequences of short-term (2-day) excess fat ingestion in a lipodystrophic (A-ZIP/F-1) mouse model in which adipose capacity is severely restricted.

Results: In wildtype littermates, this acute exposure to high fat diets resulted in excess energy intake and weight gain without any significant changes in macronutrient oxidation rates, glucose, TAG, or insulin levels. In contrast, hyperphagic lipodystrophic mice failed to gain weight; rather, they significantly increased hepatic steatosis and fat oxidation. This response was associated with a significant increase in hyperglycemia, hyperinsulinemia, glucosuria, hypertriglyceridemia, and worsening insulin tolerance.

Conclusions: These data suggest that when adipose storage reserves are saturated, excess fat intake necessarily increases fat oxidation and induces oxidative substrate competition which exacerbates insulin resistance resolving any residual energy surplus through excretion of glucose.

Objective: The skeleton, which is strongly controlled by endocrine factors, has recently been shown to also play an active endocrine role itself, specifically influencing energy metabolism. However, much less is known about this role. Therefore, we sought to identify novel endocrine factors involved in the regulation of both bone mass and whole-body glucose homeostasis.

Methods: We used transcriptomic and proteomic analysis of Y1 receptor deficient osteoblasts combined with the generation of a novel osteoglycin deficient mouse model and performed comprehensive in vivo phenotype profiling, combined with osteoglycin administration in wildtype mice and human studies.

Results: Here we identify a novel role for osteoglycin, a secreted proteoglycan, in coordinating bone accretion with changes in energy balance. Using an osteoglycin knockout mouse model, we show that at a whole body level, osteoglycin acts to suppress bone formation and modulate whole body energy supplies by altering glucose uptake through changes in insulin secretion and sensitivity, as well as by altering food intake through central signaling. Examining humans following gastric surgery as a model of negative energy balance, we show that osteoglycin is associated with BMI and lean mass as well as changes in weight, BMI, and glucose levels.

Conclusions: Thus, we identify osteoglycin as a novel factor involved in the regulation of energy homeostasis and identify a role for it in facilitating the matching of bone acquisition to alterations in energy status.

Objective: To signal, FGF19 and FGF21 require co-receptor βKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists.

Methods: We employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice.

Results: C-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice.

Conclusions: We report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.

Objective: Non-alcoholic fatty liver (NAFL) associated with obesity is a major cause of liver diseases which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF21 deficiency on liver pathology in mice consuming a conventional high fat, high sucrose (HFHS) obesogenic diet for up to 52 weeks.

Methods: C57BL6 WT and FGF21 KO mice were fed a conventional obesogenic diet and were evaluated at 16 and 52 weeks. Evaluation included metabolic assessment, liver pathology, and transcriptomic analysis.

Results: With consumption of HFHS diet, FGF21 deficient mice (FGF21 KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF21 KO mice show significantly worse fibrosis and 78% of mice develop HCC; in contrast only 6% of WT mice develop HCC. Well differentiated hepatocellular carcinomas in FGF21 KO mice were characterized by expanded hepatic plates, loss of reticulin network, cytologic atypia, and positive immunostaining for glutamine synthetase. Microarray analysis reveals enrichment of several fibroblast growth factor signaling pathways in the tumors.

Conclusions: In addition to attenuating inflammation and fibrosis in mice under a number of dietary challenges, we show here that FGF21 is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF21 in response to the high fat, high sucrose obesogenic diet may play an important role in limiting progression of liver pathology from NAFL to HCC.

Objective: Glycemic control by medical treatment represents one therapeutic strategy for diabetic patients. The Na+-d-glucose cotransporter 1 (SGLT1) is currently of high interest in this context. SGLT1 is known to mediate glucose absorption and incretin secretion in the small intestine. Recently, inhibition of SGLT1 function was shown to improve postprandial hyperglycemia. In view of the lately demonstrated SGLT1 expression in pancreatic islets, we investigated if loss of SGLT1 affects islet morphology and function.

Methods: Effects associated with the loss of SGLT1 on pancreatic islet (cyto) morphology and function were investigated by analyzing islets of a SGLT1 knockout mouse model, that were fed a glucose-deficient, fat-enriched diet (SGLT1^−/−-GDFE) to circumvent the glucose-galactose malabsorption syndrome. To distinguish diet- and Sglt1^−/−-dependent effects, wildtype mice on either standard chow (WT-SC) or the glucose-free, fat-enriched diet (WT-GDFE) were used as controls. Feeding a glucose-deficient, fat-enriched diet further required the analysis of intestinal SGLT1 expression and function under diet-conditions.

Results: Consistent with literature, our data provide evidence that small intestinal SGLT1 mRNA expression and function is regulated by nutrition. In contrast, pancreatic SGLT1 mRNA levels were not affected by the applied diet, suggesting different regulatory mechanisms for SGLT1 in diverse tissues. Morphological changes such as increased islet sizes and cell numbers associated with changes in proliferation and apoptosis and alterations of the β- and α-cell population are specifically observed for pancreatic islets of SGLT1^−/−-GDFE mice. Glucose stimulation revealed no insulin response in SGLT1^−/−-GDFE mice while WT-GDFE mice displayed only a minor increase of blood insulin. Irregular glucagon responses were observed for both, SGLT1^−/−-GDFE and WT-GDFE mice. Further, both animal groups showed a sustained release of GLP-1 compared to WT-SC controls.

Conclusions: Loss or impairment of SGLT1 results in abnormal pancreatic islet (cyto)morphology and disturbed islet function regarding the insulin or glucagon release capacity from β- or α-cells, respectively. Consequently, our findings propose a new, additional role for SGLT1 maintaining proper islet structure and function.

Objective: The inability of leptin to suppress food intake in diet-induced obesity, sometimes referred to as leptin resistance, is associated with several distinct pathological hallmarks. One prevailing theory is that impaired transport of leptin across the blood–brain barrier (BBB) represents a molecular mechanism that triggers this phenomenon. Recent evidence, however, has challenged this notion, suggesting that leptin BBB transport is acquired during leptin resistance.

Methods: To resolve this debate, we utilized a novel cerebral Open Flow Microperfusion (cOFM) method to examine leptin BBB transport in male C57BL/6J mice, fed a chow diet or high fat diet (HFD) for 20 days.

Results: Basal plasma leptin levels were 3.8-fold higher in HFD-fed mice (p < 0.05). Leptin administration (2.5 mg/kg) elicited similar pharmacokinetic profiles of circulating leptin. However, while leptin reduced food intake by 20% over 22 h in chow-fed mice, it did not affect food intake in HFD-fed mice. In spite of this striking functional difference, hypothalamic leptin levels, as measured by cOFM, did not differ between chow-fed mice and HFD-fed mice following leptin administration.

Conclusions: These data suggest that leptin transport across the BBB is not impaired in non-obese leptin resistant mice and thus unlikely to play a direct role in the progression of pharmacological leptin resistance.

Objective: Melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH) regulate food intake and body weight, glucose metabolism and convey the reward value of sucrose. In this report, we set out to establish the respective roles of MCH and conventional neurotransmitters in these neurons.

Methods: MCH neurons were profiled using Cre-dependent molecular profiling technologies (vTRAP). MCHCre mice crossed to Vglut2^fl/flmice or to DTR^fl/flwere used to identify the role of glutamate in MCH neurons. We assessed metabolic parameters such as body composition, glucose tolerance, or sucrose preference.

Results: We found that nearly all MCH neurons in the LH are glutamatergic and that a loss of glutamatergic signaling from MCH neurons from a glutamate transporter (VGlut2) knockout leads to a reduced weight, hypophagia and hyperkinetic behavior with improved glucose tolerance and a loss of sucrose preference. These effects are indistinguishable from those seen after ablation of MCH neurons. These findings are in contrast to those seen in mice with a knockout of the MCH neuropeptide, which show normal glucose preference and do not have improved glucose tolerance.

Conclusions: Overall, these data show that the vast majority of MCH neurons are glutamatergic, and that glutamate and MCH signaling mediate partially overlapping functions by these neurons, presumably by activating partially overlapping postsynaptic populations. The diverse functional effects of MCH neurons are thus mediated by a composite of glutamate and MCH signaling.