The role of adipose tissue dysfunction in hepatic insulin resistance and T2D.

The root cause of type 2 diabetes (T2D) is insulin resistance (IR), defined by the failure of cells to respond to circulating insulin to maintain lipid and glucose homeostasis. While the causes of whole-body insulin resistance are multifactorial, a major contributing factor is dysregulation of liver and adipose tissue function. Adipose dysfunction, particularly adipose tissue-IR (adipo-IR), plays a crucial role in the development of hepatic insulin resistance and the progression of metabolic dysfunction-associated steatotic liver disease (MASLD) in the context of T2D. In this review, we will focus on molecular mechanisms of hepatic insulin resistance and its association with adipose tissue function. A deeper understanding of the pathophysiological mechanisms of the transition from a healthy state to insulin resistance, impaired glucose tolerance, and T2D may enable us to prevent and intervene in the progression to T2D.

Role of human Kallistatin in glucose and energy homeostasis in mice.

OBJECTIVE: Kallistatin (KST), also known as SERPIN A4, is a circulating, broadly acting human plasma protein with pleiotropic properties. Clinical studies in humans revealed reduced KST levels in obesity. The exact role of KST in glucose and energy homeostasis in the setting of insulin resistance and type 2 diabetes is currently unknown. METHODS: Kallistatin mRNA expression in human subcutaneous white adipose tissue (sWAT) of 47 people with overweight to obesity of the clinical trial "Comparison of Low Fat and Low Carbohydrate Diets With Respect to Weight Loss and Metabolic Effects (B-SMART)" was measured. Moreover, we studied transgenic mice systemically overexpressing human KST (hKST-TG) and wild type littermate control mice (WT) under normal chow (NCD) and high-fat diet (HFD) conditions. RESULTS: In sWAT of people with overweight to obesity, KST mRNA increased after diet-induced weight loss. On NCD, we did not observe differences between hKST-TG and WT mice. Under HFD conditions, body weight, body fat and liver fat content did not differ between genotypes. Yet, during intraperitoneal glucose tolerance tests (ipGTT) insulin excursions and HOMA-IR were lower in hKST-TG (4.42 ± 0.87 AU, WT vs. 2.20 ± 0.27 AU, hKST-TG, p < 0.05). Hyperinsulinemic euglycemic clamp studies with tracer-labeled glucose infusion confirmed improved insulin sensitivity by higher glucose infusion rates in hKST-TG mice (31.5 ± 1.78 mg/kg/min, hKST-TG vs. 18.1 ± 1.67 mg/kg/min, WT, p < 0.05). Improved insulin sensitivity was driven by reduced hepatic insulin resistance (clamp hepatic glucose output: 7.7 ± 1.9 mg/kg/min, hKST-TG vs 12.2 ± 0.8 mg/kg/min, WT, p < 0.05), providing evidence for direct insulin sensitizing effects of KST for the first time. Insulin sensitivity was differentially affected in skeletal muscle and adipose tissue. Mechanistically, we observed reduced Wnt signaling in the liver but not in skeletal muscle, which may explain the effect. CONCLUSIONS: KST expression increases after weight loss in sWAT from people with obesity. Furthermore, human KST ameliorates diet-induced hepatic insulin resistance in mice, while differentially affecting skeletal muscle and adipose tissue insulin sensitivity. Thus, KST may be an interesting, yet challenging, therapeutic target for patients with obesity and insulin resistance.

Mechanisms of weight loss-induced remission in people with prediabetes: a post-hoc analysis of the randomised, controlled, multicentre Prediabetes Lifestyle Intervention Study (PLIS).

BACKGROUND: Remission of type 2 diabetes can occur as a result of weight loss and is characterised by liver fat and pancreas fat reduction and recovered insulin secretion. In this analysis, we aimed to investigate the mechanisms of weight loss- induced remission in people with prediabetes. METHODS: In this prespecified post-hoc analysis, weight loss-induced resolution of prediabetes in the randomised, controlled, multicentre Prediabetes Lifestyle Intervention Study (PLIS) was assessed, and the results were validated against participants from the Diabetes Prevention Program (DPP) study. For PLIS, between March 1, 2012, and Aug 31, 2016, participants were recruited from eight clinical study centres (including seven university hospitals) in Germany and randomly assigned to receive either a control intervention, a standard lifestyle intervention (ie, DPP-based intervention), or an intensified lifestyle intervention for 12 months. For DPP, participants were recruited from 23 clinical study centres in the USA between July 31, 1996, and May 18, 1999, and randomly assigned to receive either a standard lifestyle intervention, metformin, or placebo. In both PLIS and DPP, only participants who were randomly assigned to receive lifestyle intervention or placebo and who lost at least 5% of their bodyweight were included in this analysis. Responders were defined as people who returned to normal fasting plasma glucose (FPG; <5·6 mmol/L), normal glucose tolerance (<7·8 mmol/L), and HbA1c less than 39 mmol/mol after 12 months of lifestyle intervention or placebo or control intervention. Non-responders were defined as people who had FPG, 2 h glucose, or HbA1c more than these thresholds. The main outcomes for this analysis were insulin sensitivity, insulin secretion, visceral adipose tissue (VAT), and intrahepatic lipid content (IHL) and were evaluated via linear mixed models. FINDINGS: Of 1160 participants recruited to PLIS, 298 (25·7%) had weight loss of 5% or more of their bodyweight at baseline. 128 (43%) of 298 participants were responders and 170 (57%) were non-responders. Responders were younger than non-responders (mean age 55·6 years [SD 9·9] vs 60·4 years [8·6]; p<0·0001). The DPP validation cohort included 683 participants who lost at least 5% of their bodyweight at baseline. Of these, 132 (19%) were responders and 551 (81%) were non-responders. In PLIS, BMI reduction was similar between responders and non-responders (responders mean at baseline 32·4 kg/m2 [SD 5·6] to mean at 12 months 29·0 kg/m2 [4·9] vs non-responders 32·1 kg/m2 [5·9] to 29·2 kg/m2 [5·4]; p=0·86). However, whole-body insulin sensitivity increased more in responders than in non-responders (mean at baseline 291 mL/[min × m2], SD 60 to mean at 12 months 378 mL/[min × m2], 56 vs 278 mL/[min × m2], 62, to 323 mL/[min × m2], 66; p<0·0001), whereas insulin secretion did not differ within groups over time or between groups (responders mean at baseline 175 pmol/mmol [SD 64] to mean at 12 months 163·7 pmol/mmol [60·6] vs non-responders 158·0 pmol/mmol [55·6] to 154·1 pmol/mmol [56·2]; p=0·46). IHL decreased in both groups, without a difference between groups (responders mean at baseline 10·1% [SD 8·7] to mean at 12 months 3·5% [3·9] vs non-responders 10·3% [8·1] to 4·2% [4·2]; p=0·34); however, VAT decreased more in responders than in non-responders (mean at baseline 6·2 L [SD 2·9] to mean at 12 months 4·1 L [2·3] vs 5·7 L [2·3] to 4·5 L [2·2]; p=0·0003). Responders had a 73% lower risk of developing type 2 diabetes than non-responders in the 2 years after the intervention ended. INTERPRETATION: By contrast to remission of type 2 diabetes, resolution of prediabetes was characterised by an improvement in insulin sensitivity and reduced VAT. Because return to normal glucose regulation (NGR) prevents development of type 2 diabetes, we propose the concept of remission of prediabetes in analogy to type 2 diabetes. We suggest that remission of prediabetes should be the primary therapeutic aim in individuals with prediabetes. FUNDING: German Federal Ministry for Education and Research via the German Center for Diabetes Research; the Ministry of Science, Research and the Arts Baden-Württemberg; the Helmholtz Association and Helmholtz Munich; the Cluster of Excellence Controlling Microbes to Fight Infections; and the German Research Foundation.

FGF1 and insulin control lipolysis by convergent pathways.

Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.

Transcriptional refractoriness is dependent on core promoter architecture.

Genes are often transcribed in random bursts followed by long periods of inactivity. Here we employ the light-activatable white collar complex (WCC) of Neurospora to study the transcriptional bursting with a population approach. Activation of WCC by a light pulse triggers a synchronized wave of transcription from the frequency promoter followed by an extended period (∼1 h) during which the promoter is refractory towards restimulation. When challenged by a second light pulse, the newly activated WCC binds to refractory promoters and has the potential to recruit RNA polymerase II (Pol II). However, accumulation of Pol II and phosphorylation of its C-terminal domain repeats at serine 5 are impaired. Our results suggest that refractory promoters carry a physical memory of their recent transcription history. Genome-wide analysis of light-induced transcription suggests that refractoriness is rather widespread and a property of promoter architecture.

Combinatorial control of light induced chromatin remodeling and gene activation in Neurospora.

Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression.

Dawn- and dusk-phased circadian transcription rhythms coordinate anabolic and catabolic functions in Neurospora.

BACKGROUND: Circadian clocks control rhythmic expression of a large number of genes in coordination with the 24 hour day-night cycle. The mechanisms generating circadian rhythms, their amplitude and circadian phase are dependent on a transcriptional network of immense complexity. Moreover, the contribution of post-transcriptional mechanisms in generating rhythms in RNA abundance is not known. RESULTS: Here, we analyzed the clock-controlled transcriptome of Neurospora crassa together with temporal profiles of elongating RNA polymerase II. Our data indicate that transcription contributes to the rhythmic expression of the vast majority of clock-controlled genes (ccgs) in Neurospora. The ccgs accumulate in two main clusters with peak transcription and expression levels either at dawn or dusk. Dawn-phased genes are predominantly involved in catabolic and dusk-phased genes in anabolic processes, indicating a clock-controlled temporal separation of the physiology of Neurospora. Genes whose expression is strongly dependent on the core circadian activator WCC fall mainly into the dawn-phased cluster while rhythmic genes regulated by the glucose-dependent repressor CSP1 fall predominantly into the dusk-phased cluster. Surprisingly, the number of rhythmic transcripts increases about twofold in the absence of CSP1, indicating that rhythmic expression of many genes is attenuated by the activity of CSP1. CONCLUSIONS: The data indicate that the vast majority of transcript rhythms in Neurospora are generated by dawn and dusk specific transcription. Our observations suggest a substantial plasticity of the circadian transcriptome with respect to the number of rhythmic genes as well as amplitude and phase of the expression rhythms and emphasize a major role of the circadian clock in the temporal organization of metabolism and physiology.

Fibroblast Growth Factor Signaling in Metabolic Regulation.

The prevalence of obesity is a growing health problem. Obesity is strongly associated with several comorbidities, such as non-alcoholic fatty liver disease, certain cancers, insulin resistance, and type 2 diabetes, which all reduce life expectancy and life quality. Several drugs have been put forward in order to treat these diseases, but many of them have detrimental side effects. The unexpected role of the family of fibroblast growth factors in the regulation of energy metabolism provides new approaches to the treatment of metabolic diseases and offers a valuable tool to gain more insight into metabolic regulation. The known beneficial effects of FGF19 and FGF21 on metabolism, together with recently discovered similar effects of FGF1 suggest that FGFs and their derivatives carry great potential as novel therapeutics to treat metabolic conditions. To facilitate the development of new therapies with improved targeting and minimal side effects, a better understanding of the molecular mechanism of action of FGFs is needed. In this review, we will discuss what is currently known about the physiological roles of FGF signaling in tissues important for metabolic homeostasis. In addition, we will discuss current concepts regarding their pharmacological properties and effector tissues in the context of metabolic disease. Also, the recent progress in the development of FGF variants will be reviewed. Our goal is to provide a comprehensive overview of the current concepts and consensuses regarding FGF signaling in metabolic health and disease and to provide starting points for the development of FGF-based therapies against metabolic conditions.

Alteration of light-dependent gene regulation by the absence of the RCO-1/RCM-1 repressor complex in the fungus Neurospora crassa.

The activation of transcription by light in the fungus Neurospora crassa requires the White Collar Complex (WCC), a photoreceptor and transcription factor complex. After light reception two WCCs interact and bind the promoters of light-regulated genes to activate transcription. This process is regulated by VVD, a small photoreceptor that disrupts the interaction between WCCs and leads to a reduction in transcription after long exposures to light. The N. crassa RCO-1/RCM-1 repressor complex is the homolog of the Tup1-Ssn6 repressor complex in yeast, and its absence modifies photoadaptation. We show that the absence of the RCO-1/RCM-1 repressor complex leads to several alterations in transcription that are gene-specific: an increase in the accumulation of mRNAs in the dark, a repression of transcription, and a derepression of transcription after long exposures to light. The absence of the RCO-1/RCM-1 repressor complex leads to lower VVD levels that are available for the regulation of the activity of the WCC. The reduction in the amount of VVD results in increased WCC binding to the promoters of light-regulated genes in the dark and after long exposures to light, leading to the modification of photoadaptation that has been observed in rco-1 and rcm-1 mutants. Our results show that the photoadaptation phenotype of mutants in the RCO-1/RCM-1 repressor complex is, at least in part, an indirect consequence of the reduction of vvd transcription, and the resulting modification in the regulation of transcription by the WCC.

Circadian clocks and energy metabolism.

Circadian clocks orchestrate behavioral and physiological processes in a time-of-day dependent manner. The network of clock-controlled genes is intimately interconnected with metabolic regulatory circuits. Circadian clocks rhythmically regulate the expression and activity of key metabolic players, which in turn feed back on the circadian machinery on the transcriptional and post-transcriptional level. Mutations of clock genes are often associated with metabolic defects, especially in lipid and glucose metabolism. Accumulating data suggest that the reciprocal coordination of circadian and metabolic pathways is crucial for cellular homeostasis and the health of the organism.

Metabolic compensation of the Neurospora clock by a glucose-dependent feedback of the circadian repressor CSP1 on the core oscillator.

Conidial separation 1 (CSP1) is a global transcription repressor. It is expressed under control of the white collar complex (WCC), the core transcription factor of the circadian clock of Neurospora. Here we report that the length of the circadian period decreases with increasing glucose concentrations in csp1 mutant strains, while the period is compensated for changes in glucose concentration in wild-type strains. Glucose stimulated CSP1 expression. Overexpression of CSP1 caused period lengthening and, eventually, complete dampening of the clock rhythm. We show that CSP1 inhibits expression of the WHITE COLLAR 1 (WC1) subunit of the WCC by repressing the wc1 promoter. Glucose-dependent repression of wc1 transcription by CSP1 compensated for the enhanced translation of WC1 at high glucose levels, resulting in glucose-independent expression of the WCC and, hence, metabolic compensation that maintained a constant circadian period. Thus, the negative feedback of CSP1 on WC1 expression constitutes a molecular pathway that coordinates energy metabolism and the circadian clock.

Glycogen synthase kinase is a regulator of the circadian clock of Neurospora crassa.

Timekeeping by circadian clocks relies upon precise adjustment of expression levels of clock proteins. Here we identify glycogen synthase kinase (GSK) as a novel and critical component of the circadian clock of Neurospora crassa that regulates the abundance of its core transcription factor white collar complex (WCC) on a post-transcriptional level. We show that GSK specifically binds and phosphorylates both subunits of the WCC. Reduced expression of GSK promotes an increased accumulation of WC-1, the limiting factor of the WCC, causing an acceleration of the circadian clock and a shorter free-running period.

A global circadian repressor controls antiphasic expression of metabolic genes in Neurospora.

The white-collar complex (WCC), the core transcription factor of the circadian clock of Neurospora, activates morning-specific expression of the transcription repressor CSP1. Newly synthesized CSP1 exists in a transient complex with the corepressor RCM1/RCO1 and the ubiquitin ligase UBR1. CSP1 is rapidly hyperphosphorylated and degraded via UBR1 and its ubiquitin conjugase RAD6. Genes controlled by CSP1 are rhythmically expressed and peak in the evening (i.e., in antiphase to morning-specific genes directly controlled by WCC). Rhythmic expression of these second-tier genes depends crucially on phosphorylation and rapid turnover of CSP1, which ensures tight coupling of CSP1 abundance and function to the circadian activity of WCC. Negative feedback of CSP1 on its own transcription buffers the amplitude of CSP1-dependent oscillations against fluctuations of WCC activity. CSP1 predominantly regulates genes involved in metabolism. It controls ergosterol synthesis and fatty acid desaturases and thereby modulates the lipid composition of membranes.

Transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Activity of the circadian transcription factor White Collar Complex is modulated by phosphorylation of SP-motifs.

Posttranslational modifications, particularly phosphorylation, regulate activity, stability and localization of proteins in circadian clocks, thereby contributing to a stable oscillation with a period of approximately 24h. The White Collar Complex (WCC) is the central transcription factor of the circadian clock of Neurospora crassa. Its activity is regulated in a circadian manner by rhythmic phosphorylation, mediated by the clock protein Frequency (FRQ). Here we present purification of TAP-tagged WCC and identification of novel phosphorylation sites of WC-1 and WC-2, all of which appear to be proline directed. Exchange of a single WC-2 serine residue (S433) to alanine or aspartate affects WCC-dependent transcription and circadian period, suggesting an important role of WC-2 S433 phosphorylation for WCC activity and circadian timing.