OBJECTIVE:Though Tcf7l2 harbors the strongest genetic association with diabetes identified thus far, how it promotes metabolic disease remains unclear. Our aim was to dissect the contribution of hepatic TCF7L2. METHODS:Mice with liver-specific knockout of Tcf7l2 produced by targeted deletion of exon 1 were subjected to physiological characterization, single nucleus sequencing, and metabolite profiling. In parallel, a phenome-wide association study was performed in humans. RESULTS:We found that liver-specific deletion of Tcf7l2 had little effect on plasma glucose, but disrupted hepatic zonation. That is, many genes normally show gradients of expression across the liver lobule; in the absence of Tcf7l2, these gradients collapsed. One major consequence was the disorganization of glutamine metabolism, with a loss of the glutamine production program, ectopic expression of the glutamine consumption program, and a decrease in glutamine levels. In parallel, glutamine was found to be the most significantly decreased metabolite in the plasma of individuals harboring the rs7903146 variant in Tcf7l2. CONCLUSIONS:Taken together, these data indicate that hepatic TCF7L2 has a secondary role in glycemic control, but a primary role in maintaining transcriptional architecture and glutamine homeostasis.

Obesity's metabolic impact goes beyond peripheral insulin resistance, influencing the brain and thereby disrupting energy homeostasis by altering anabolic and catabolic reactions. Density-enhanced phosphatase-1 (DEP-1), a ubiquitously expressed receptor-like tyrosine phosphatase, has emerged as a novel regulator with a specific role in dephosphorylating the insulin receptor (IR). Strikingly, diet-induced obese mice exhibit elevated DEP-1 expression in metabolically sensitive tissues. In this study, we sought to elucidate the role of DEP-1 in brain insulin signaling and highlight its potential impact on the central regulation of metabolism in vitro and in vivo. To explore DEP-1 deficiency in vitro, CRISPR/Cas9 was employed to create a DEP-1 knockout (KO) in mouse Neuro-2a cells. Given DEP-1’s high expression in the forebrain and the region's abundant IR expression, we examined the metabolic consequences of DEP-1 deficiency in the forebrain by deleting Dep-1 using CamKIIa Cre mice (DEFO KO). DEP-1 KO cells exhibited an increase in IR phosphorylation and downstream signaling upon acute insulin stimulation, coupled with unexpected activation of AMP-activated protein kinase (AMPK) cascade. Similarly, male DEFO KO mice showed heightened insulin and AMPK signaling in the forebrain under random-fed conditions. A notable outcome manifested in the gonadal white adipose tissue of DEFO KO mice, where an ex vivo lipolysis assay revealed elevated basal lipolytic potential compared to control mice stimulated with isoproterenol, indicating enhanced sympathetic activation. Brown adipose tissue (BAT) of DEFO KO mice also demonstrated in a mitochondrial respiration assay, an upregulation of fatty acid oxidation, signifying increased BAT activity. Both tissues also revealed higher beta-adrenergic receptor gene expression, suggestive of sympathetic activity. In conclusion, DEP-1 is a pivotal neuronal IR phosphatase, that prevents the establishment of futile cycles and emerges as a novel modulator of energy metabolism. Disclosure S. Chopra: None. O.L.J. Kadiri: None. J. Ulke: None. R. Hauffe: None. W. Jonas: None. C.A. Bishop: None. S. Cheshmeh: None. M. Rath: None. M. Schell: None. K. Kappert: None. A. Kleinridders: Speaker's Bureau; Novo Nordisk A/S, Daiichi Sankyo. Funding German Research Foundation (DFG)KL2399/6-1

Obesity is associated with insulin and leptin resistance, which affects peripheral tissues as well as the brain. The novel density-enhanced phosphatase-1 (DEP-1), is a known negative modulator of insulin and leptin signaling. Intriguingly, diet-induced obese mice have increased DEP-1 expression in metabolic sensitive tissues, including the brain. In this study, the regulation of DEP-1-dependent metabolic effects was investigated in vitro and in vivo.

A healthy metabolism relies on precise regulation of anabolic and catabolic pathways. While insulin deficiency impairs anabolism, insulin resistance in obesity causes metabolic dysfunction, especially via altered brain insulin receptor (IR) activity. Density-enhanced phosphatase 1 (DEP-1) negatively modulates the IR in peripheral tissues. Our study shows that DEP-1 is an insulin-regulated gene, dysregulated in obesity, and uncovers its role in brain insulin signaling, impacting both anabolic and catabolic pathways. Neuro-2a cells lacking DEP-1 demonstrated heightened IR phosphorylation upon acute insulin stimulation. This coincided with simultaneous AMP-activated protein kinase (AMPK) activation, which governs catabolic pathways, due to increased phospholipase C-gamma 1 signaling. These opposing pathways in male DEP-1 forebrain-specific knockout mice resulted in elevated lipolysis in white adipose tissue and fat oxidation in brown adipose tissue, with enhanced sympathetic activation and β-adrenergic receptor expression. In conclusion, DEP-1 deficiency causes the simultaneous activation of IR and AMPK signaling in the brain, with enhanced sympathetic activity in adipose tissues.

BACKGROUND: Patients with diabetes exhibit an increased prevalence for emotional disorders compared with healthy humans, partially due to a shared pathogenesis including hormone resistance and inflammation, which is also linked to intestinal dysbiosis. The preventive intake of probiotic lactobacilli has been shown to improve dysbiosis along with mood and metabolism. Yet, a potential role of Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus 0030) (LR) in improving emotional behavior in established obesity and the underlying mechanisms are unknown.METHODS: Female and male C57BL/6N mice were fed a low-fat diet (10% kcal from fat) or high-fat diet (HFD) (45% kcal from fat) for 6 weeks, followed by daily oral gavage of vehicle or 1 3 108 colony-forming units of LR, and assessment of anxiety-and depressive-like behavior. Cecal microbiota composition was analyzed using 16S ribosomal RNA sequencing, plasma and cerebrospinal fluid were collected for metabolomic analysis, and gene expression of different brain areas was assessed using reverse transcriptase quantitative polymerase chain reaction.RESULTS: We observed that 12 weeks of HFD feeding induced hyperinsulinemia, which was attenuated by LR application only in female mice. On the contrary, HFD-fed male mice exhibited increased anxiety-and depressive-like behavior, where the latter was specifically attenuated by LR application, which was independent of metabolic changes. Furthermore, LR application restored the HFD-induced decrease of tyrosine hydroxylase, along with normalizing cholecystokinin gene expression in dopaminergic brain regions; both tyrosine hydroxylase and cholecystokinin are involved in signaling pathways impacting emotional disorders.CONCLUSIONS: Our data show that LR attenuates depressive-like behavior after established obesity, with changes in the dopaminergic system in male mice, and mitigates hyperinsulinemia in obese female mice.

Type-2 Diabetes (T2D) is characterized by insulin resistance and accompanied by psychiatric comorbidities including major depressive disorders (MDD). Patients with T2D are twice more likely to suffer from MDD and clinical studies have shown that insulin resistance is positively correlated with the severity of depressive symptoms. However, the potential contribution of central insulin signaling in MDD in patients with T2D remains elusive. Here we hypothesized that insulin modulates the serotonergic (5-HT) system to control emotional behavior and that insulin resistance in 5-HT neurons contributes to the development of mood disorders in T2D. Our results show that insulin directly modulates the activity of dorsal raphe (DR) 5-HT neurons to dampen 5-HT neurotransmission through a 5-HT1A receptor-mediated inhibitory feedback. In addition, insulin-induced 5-HT neuromodulation is necessary to promote anxiolytic-like effect in response to intranasal insulin delivery. Interestingly, such an anxiolytic effect of intranasal insulin as well as the response of DR 5-HT neurons to insulin are both blunted in high-fat diet-fed T2D animals. Altogether, these findings point to a novel mechanism by which insulin directly modulates the activity of DR 5-HT neurons to dampen 5-HT neurotransmission and control emotional behaviors, and emphasize the idea that impaired insulin-sensitivity in these neurons is critical for the development of T2D-associated mood disorders.

Prevalence of depression is higher in diabetic patients compared to healthy humans due to a shared pathogenesis including insulin resistance and inflammation. These factors are also linked to intestinal dysbiosis. Interestingly, preventive intake of probiotic lactobacilli has been shown to improve dysbiosis along with mood and metabolism. Yet, a potential therapeutic role and underlying mechanism of Lactobacillus rhamnosus GG (LGG, ATCC 53103) to improve emotional behavior in diet-induced obesity is unknown. Male C57BL/6N mice were fed a low-fat diet (LFD, 10% kcal from fat) or high-fat diet (HFD, 45% kcal from fat) for 6 weeks and followed by daily oral gavage of vehicle or 1x108 CFU of LGG. Dark-Light Box Test was used to evaluate anxiety, Mousetail Suspension Test (MST) and Splash Test were used to assess depressive-like behavior and motivation for self-care. Cecal microbiome composition was analyzed using 16S rRNA Sequencing. Plasma and CSF were collected for metabolomic analysis, and gene expression was analyzed using qPCR. We observed that 12 weeks of HFD feeding increased anxiety and depressive-like behavior. Strikingly, LGG decreased specifically depressive-like behavior in the MST which was confirmed by the Splash Test. Metagenomics analysis revealed that HFD but not LGG alters cecal microbiome composition, indicating a transient effect. Moreover, HFD feeding significantly altered plasma lipid metabolism whereas LGG decreased branched chain amino acids which associated with improved emotional behavior. In dopaminergic brain regions, LGG restored the HFD-induced decrease of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, while not altering enzymes responsible for dopamine degradation. Our data indicate that LGG attenuates specifically depressive-like behavior in HFD-fed mice with a molecular signature of restored dopamine synthesis. Disclosure M.Schell: Other Relationship; Novozymes A/S, Denmark. K.Wardelmann: None. R.Hauffe: None. M.Rath: None. S.Chopra: None. A.Kleinridders: Other Relationship; Novozymes. Funding German Ministry of Education and Research (BMBF) and the State of Brandenburg (DZD grant 82DZD00302 and BMBF grant 031B0569) .

The regulation of energy homeostasis is controlled by the brain and, besides requiring high amounts of energy, it relies on functional insulin/insulin-like growth factor (IGF)-1 signalling in the central nervous system. This energy is mainly provided by mitochondria in form of ATP. Thus, there is an intricate interplay between mitochondrial function and insulin/IGF-1 action to enable functional brain signalling and, accordingly, propagate a healthy metabolism. To adapt to different nutritional conditions, the brain is able to sense the current energy status via mitochondrial and insulin signalling-dependent pathways and exerts an appropriate metabolic response. However, regional, cell type and receptor-specific consequences of this interaction occur and are linked to diverse outcomes such as altered nutrient sensing, body weight regulation or even cognitive function. Impairments of this cross-talk can lead to obesity and glucose intolerance and are linked to neurodegenerative diseases, yet they also induce a self-sustainable, dysfunctional 'metabolic triangle' characterised by insulin resistance, mitochondrial dysfunction and inflammation in the brain. The identification of causal factors deteriorating insulin action, mitochondrial function and concomitantly a signature of metabolic stress in the brain is of utter importance to offer novel mechanistic insights into development of the continuously rising prevalence of non-communicable diseases such as type 2 diabetes and neurodegeneration. This review aims to determine the effect of insulin action on brain mitochondrial function and energy metabolism. It precisely outlines the interaction and differences between insulin action, insulin-like growth factor (IGF)-1 signalling and mitochondrial function; distinguishes between causality and association; and reveals its consequences for metabolism and cognition. We hypothesise that an improvement of at least one signalling pathway can overcome the vicious cycle of a self-perpetuating metabolic dysfunction in the brain present in metabolic and neurodegenerative diseases.

The prevalence of depression and associated mood disorders is higher in diabetic patients compared to healthy humans. Contributing factors might be deteriorated dopamine signaling, increased inflammation or an overactivation of the hypothalamic-pituitary-adrenal axis. These factors are also linked to an intestinal dysbiosis. Interestingly, preventive intake of probiotic lactobacilli to improve dysbiosis has been shown to improve mood and metabolism. Yet, the potential, therapeutic role of Lactobacillus rhamnosus ATCC 53103 (53103) to improve emotional behavior in diet-induced obese conditions is unknown and was investigated in this study.

OBJECTIVE:Insulin regulates mitochondrial function, thereby propagating an efficient metabolism. Conversely, diabetes and insulin resistance are linked to mitochondrial dysfunction with a decreased expression of the mitochondrial chaperone HSP60. The aim of this investigation was to determine the effect of a reduced HSP60 expression on the development of obesity and insulin resistance. METHODS:Control and heterozygous whole-body HSP60 knockout (Hsp60+/-) mice were fed a high-fat diet (HFD, 60% calories from fat) for 16 weeks and subjected to extensive metabolic phenotyping. To understand the effect of HSP60 on white adipose tissue, microarray analysis of gonadal WAT was performed, ex vivo experiments were performed, and a lentiviral knockdown of HSP60 in 3T3-L1 cells was conducted to gain detailed insights into the effect of reduced HSP60 levels on adipocyte homeostasis. RESULTS:Male Hsp60+/- mice exhibited lower body weight with lower fat mass. These mice exhibited improved insulin sensitivity compared to control, as assessed by Matsuda Index and HOMA-IR. Accordingly, insulin levels were significantly reduced in Hsp60+/- mice in a glucose tolerance test. However, Hsp60+/- mice exhibited an altered adipose tissue metabolism with elevated insulin-independent glucose uptake, adipocyte hyperplasia in the presence of mitochondrial dysfunction, altered autophagy, and local insulin resistance. CONCLUSIONS:We discovered that the reduction of HSP60 in mice predominantly affects adipose tissue homeostasis, leading to beneficial alterations in body weight, body composition, and adipocyte morphology, albeit exhibiting local insulin resistance.

Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.

Overconsumption of high-fat and cholesterol-containing diets is detrimental for metabolism and mitochondrial function, causes inflammatory responses and impairs insulin action in peripheral tissues. Dietary fatty acids can enter the brain to mediate the nutritional status, but also to influence neuronal homeostasis. Yet, it is unclear whether cholesterol-containing high-fat diets (HFDs) with different combinations of fatty acids exert metabolic stress and impact mitochondrial function in the brain. To investigate whether cholesterol in combination with different fatty acids impacts neuronal metabolism and mitochondrial function, C57BL/6J mice received different cholesterol-containing diets with either high concentrations of long-chain saturated fatty acids or soybean oil-derived poly-unsaturated fatty acids. In addition, CLU183 neurons were stimulated with combinations of palmitate, linoleic acid and cholesterol to assess their effects on metabolic stress, mitochondrial function and insulin action. The dietary interventions resulted in a molecular signature of metabolic stress in the hypothalamus with decreased expression of occludin and subunits of mitochondrial electron chain complexes, elevated protein carbonylation, as well as c-Jun N-terminal kinase (JNK) activation. Palmitate caused mitochondrial dysfunction, oxidative stress, insulin and insulin-like growth factor-1 (IGF-1) resistance, while cholesterol and linoleic acid did not cause functional alterations. Finally, we defined insulin receptor as a novel negative regulator of metabolically stress-induced JNK activation.

Background: INDY (I’m Not Dead Yet) is a plasma membrane citrate transporter and is highly expressed in liver and brain. In mammals, whole body deletion of the coding gene (mIndy, Slc13a5) increased energy expenditure and protected mice from diet-induced obesity and insulin resistance. Generation of neuronal mIndy-KO (NINKO) mice revealed improved insulin sensitivity (IS) in these mice, mediated through better hepatic IS. Gene expression studies in wildtype C57Bl/6 mice revealed high expression of mIndy in the hypothalamus (HTM), which is the main brain area regulating hepatic glucose metabolism. One mechanisms is the regulation through AMPK. Reduced hypothalamic AMPK phosphorylation is known to reduce hepatic glucose output, probably mediated via vagal nerve signaling. Hypothesis: Hepatic glucose production is regulated via mIndy expression in the HTM. Methods: Hyperinsulinemic-euglycemic clamp studies were performed to assess IS after hepatic vagotomy of NINKO mice. NestinCre+ controls were sham-denervated. Ex vivo brain slices of C57Bl/6 mice and the neuronal hypothalamic cell line CLU183 were acutely stimulated with 1 mM sodium citrate to investigate the effect of citrate on the brain and neurons. Results: Acute stimulation of brain slices and CLU183 cells with citrate decreased cellular respiration (-19.7%, p≤0.01), ATP production (-37.7%, p≤0.01) and increased AMPK phosphorylation (+24.7%, p≤0.001). In line with that, AMPK phosphorylation in the HTM of NINKO mice was significantly reduced (-68.1%, p≤0.05). With hepatic branch vagotomy, no differences in hepatic IS could be observed anymore (suppression of basal hepatic glucose output (%); NestinCre+: 100.0±27.2, NINKO: 99.4±8.8). Conclusion: These data suggest that neuronal mIndy is a critical regulator of glucose homeostasis in mammals, probably regulated via AMPK phosphorylation in the HTM and vagal nerve signaling. Further studies will address the exact mechanisms involved in the effect. Disclosure A. Kurzbach: None. M. Schell: None. D.M. Willmes: None. N. El-Agroudy: None. A. Kleinridders: None. A.L. Birkenfeld: None.

Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of patients with diabetes are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of IR with a reduction of mRNA expression of selenoproteins Txnrd3, Sephs2, and Gpx3 in gonadal adipose tissue. Consistently, GPX3 is also decreased in adipose tissue of insulin-resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation, and elevated IR expression in WAT. Again, IR expression correlated positively with Gpx3 expression, a phenotype that is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduced IR expression and insulin sensitivity in 3T3-L1 preadipocytes. Overall, our data identify GPx3 as a potentially novel regulator of IR expression and insulin sensitivity in adipose tissue.

Objective: Insulin action in the brain controls metabolism and brain function, which is linked to proper mitochondrial function. Conversely, brain insulin resistance associates with mitochondrial stress and metabolic and neurodegenerative diseases. In the present study, we aimed to decipher the impact of hypothalamic insulin action on mitochondrial stress responses, function and metabolism. Methods: To investigate the crosstalk of insulin action and mitochondrial stress responses (MSR), namely the mitochondrial unfolded protein response (UPRmt) and integrated stress response (ISR), qPCR, western blotting, and mitochondrial activity assays were performed. These methods were used to analyze the hypothalamic cell line CLU183 treated with insulin in the presence or absence of the insulin receptor as well as in mice fed a high fat diet (HFD) for three days and STZ-treated mice without or with insulin therapy. Intranasal insulin treatment was used to investigate the effect of acute brain insulin action on metabolism and mitochondrial stress responses. Results: Acute HFD feeding reduces hypothalamic mitochondrial stress responsive gene expression of Atf4, Chop, Hsp60, Hsp10, ClpP, and Lonp1 in C57BL/6N mice. We show that insulin via ERK activation increases the expression of MSR genes in vitro as well as in the hypothalamus of streptozotocin-treated mice. This regulation propagates mitochondrial function by controlling mitochondrial proteostasis and prevents excessive autophagy under serum deprivation. Finally, short-term intranasal insulin treatment activates MSR gene expression in the hypothalamus of HFD-fed C57BL/6N mice and reduces food intake and body weight development. Conclusions: We define hypothalamic insulin action as a novel master regulator of MSR, ensuring proper mitochondrial function by controlling mitochondrial proteostasis and regulating metabolism. (C) 2019 The Authors. Published by Elsevier GmbH.

The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner. Furthermore, interventions to reduce TMAO, either by manipulation of the gut microbiota or by inhibition of the TMAO synthesizing enzyme, flavin-containing monooxygenase 3, can reduce PERK activation and FoxO1 levels in the liver. Taken together, these data suggest TMAO and PERK may be central to the pathogenesis of the metabolic syndrome.

BACKGROUND/AIM:Inadequate fluid intake is assumed to be a trigger of water-loss dehydration, which is a major health risk in aged and geriatric populations. Thus, there is a need to search for easy to use diagnostic tests to identify dehydration. Our overall aim was to investigate whether skin barrier parameters could be used for predicting fluid intake and/or hydration status in geriatric patients.METHODS:An explorative observational comparative study was conducted in a geriatric hospital including patients aged 65 years and older. We measured 3-day fluid intake, skin barrier parameters, Overall Dry Skin Score, serum osmolality, cognitive and functional health, and medications.RESULTS:Forty patients were included (mean age 78.45 years and 65% women) with a mean fluid intake of 1,747 mL/day. 20% of the patients were dehydrated and 22.5% had an impending dehydration according to serum osmolality. Multivariate analysis suggested that skin surface pH and epidermal hydration at the face were associated with fluid intake. Serum osmolality was associated with epidermal hydration at the leg and skin surface pH at the face. Fluid intake was not correlated with serum osmolality. Diuretics were associated with high serum osmolality.CONCLUSIONS:Approximately half of the patients were diagnosed as being dehydrated according to osmolality, which is the current reference standard. However, there was no association with fluid intake, questioning the clinical relevance of this measure. Results indicate that single skin barrier parameters are poor markers for fluid intake or osmolality. Epidermal hydration might play a role but most probably in combination with other tests.

Citrate, one of the most abundant metabolites in the cerebrospinal fluid (CSF), is a potent chelator and important for neuronal activity. Decreased blood citrate levels were found in diabetic humans. Global deletion of the citrate transporter SLC13A5 increases plasma citrate levels, activates hepatic AMP-activated protein kinase (AMPK) and protects against obesity and insulin resistance, suggesting that increased extracellular citrate levels might be beneficial. Yet, the role of extracellular citrate on brain function is not well understood and was investigated in the current study.