American Journal of Physiology-Endocrinology and Metabolism

The acceleration of hepatic lipid disposal during acute exercise has been proposed as a contributor to the anti-steatotic effects of exercise training. Ketogenesis, which produces acetoacetate (AcAc) and {beta}-hydroxybutyrate ({beta}OHB) from fatty acids, is among the lipid disposal pathways stimulated by exercise. This study tested the hypothesis that hepatic ketogenesis is necessary for exercise training to lower liver lipids. Liver-specific 3-hydroxymethylglutaryl-CoA synthase 2 knockout (HMGCS2 KO) mice and wild type (WT) littermates underwent sedentary, acute exercise, and exercise training protocols. Liver ketone bodies and lipids were determined via mass spectrometry platforms. Stable isotope infusions in conscious, unrestrained mice defined mitochondrial oxidative fluxes at rest and during exercise. Loss of hepatic HMGCS2 decreased liver AcAc and {beta}OHB concentrations and impaired their increase during exercise. Liver triacylglycerides (TAGs) were comparable between genotypes at rest (i.e., ad libitum fed and short fasted conditions). In contrast, liver TAGs were elevated in HMGCS2 KO mice following acute, non-exhaustive exercise. Liver TCA cycle flux was higher in KO mice at rest. During exercise, TCA cycle flux increased in both WT and KO mice but was not different between genotypes with greater exercise duration. This suggests that enhanced disposal of lipids via the TCA cycle may prevent liver lipid accumulation in HMGCS2 KO mice under sedentary conditions, but not during exercise. Unexpectedly, exercise training decreased liver TAGs similarly in both HMGCS2 KO and WT mice. In conclusion, hepatic ketogenesis supports liver lipid homeostasis during acute exercise, but is not required for exercise training to lower liver lipids. NEW & NOTEWORTHYExercise training has been proposed to mitigate liver steatosis partly through enhanced hepatic lipid disposal. During acute exercise, the disposal of fatty acids to ketone bodies is stimulated. This study tested the hypothesis that hepatic ketogenesis was required for exercise training to reduce liver fat in mice. The results show that hepatic ketogenesis is needed to prevent lipid accumulation during acute exercise, but is not necessary for exercise training to lower liver lipids.

The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via de novo lipogenesis (DNL) when carbohydrates are abundant. Mitochondrial pyruvate metabolism is thought to play important roles in both gluconeogenesis and DNL. We examined the effects of hepatocyte-specific mitochondrial pyruvate carrier (MPC) deletion on the fasting-refeeding response. Rates of DNL during refeeding were impaired by liver MPC deletion, but this did not reduce intrahepatic lipid content. During fasting, glycerol is converted to glucose by two pathways; a direct cytosolic pathway essentially reversing glycolysis and an indirect mitochondrial pathway requiring the MPC. MPC deletion reduced the incorporation of 13C-glycerol into TCA cycle metabolites but not into newly synthesized glucose. However, suppression of glycerol metabolism did not affect glucose concentrations in fasted hepatocyte-specific MPC-deficient mice. Thus, glucose production by kidney and intestine may compensate for MPC deficiency in hepatocytes.

The second-meal phenomenon refers to the improved glycemic response to a subsequent identical meal. Postprandial net hepatic glucose uptake (NHGU) is governed by the combined effects of three regulatory factors: insulin action (IA), initiated by hyperinsulinemia; glucose effectiveness (GE), driven by hyperglycemia; and the portal glucose signal (PGS), activated by glucose delivery into the hepatoportal circulation. Previous studies demonstrated that morning (AM) hyperinsulinemia primes the liver, causing substantially enhanced NHGU later in the day; however, it remained unclear which component of the afternoon (PM) response is augmented. To address this, we assessed how AM insulin elevation influences PM IA, GE, and the PGS. Dogs underwent an AM clamp with either a 4h hyperinsulinemic prime (Prime, n=8) or basal insulin delivery (No Prime, n=8). After a 1.5-hour rest, both groups underwent a PM hyperglycemic clamp (with portal glucose delivery) under basal insulin conditions. During the PM clamp, NHGU was significantly greater in the Prime versus No Prime group (2.2{+/-}0.3 vs. 0.1{+/-}0.3 mg/kg/min, P<0.005), indicating priming enhanced GE and/or PGS effects. In prior experiments with all three stimuli present in the PM (IA, GE, and PGS), AM insulin priming increased PM NHGU by 3.8 mg/kg/min. Thus, while AM insulin priming alone enhanced GE and/or PGS, the full effect requires an elevation in PM insulin, suggesting that morning insulin exposure primes the liver by augmenting both afternoon insulin action and glucose action.

Glucose tolerance improves significantly upon consuming a second, identical meal later in the day (second meal phenomenon). We previously established that morning hyperinsulinemia primes the liver for increased afternoon hepatic glucose uptake (HGU). Although the route of insulin delivery is an important determinant of the mechanisms by which insulin regulates liver glucose metabolism (direct hepatic vs indirect insulin action), it is not known if insulins delivery route affects the second meal response. To determine whether morning peripheral insulin delivery (as occurs clinically (subcutaneous)) can enhance afternoon HGU, conscious dogs were treated in the morning with insulin delivered via the portal vein, or peripherally (leg vein), while glucose was infused to maintain euglycemia. Consequently, arterial insulin levels increased similarly in both groups, but relative hepatic insulin deficiency occurred when insulin was delivered peripherally. In the afternoon, all animals were challenged with the same hyperinsulinemic-hyperglycemic clamp to simulate identical postprandial-like conditions. The substantial enhancement of HGU in the afternoon caused by morning portal vein insulin delivery was lost when insulin was delivered peripherally. This indicates that morning insulin does not cause the second meal phenomenon via its indirect actions on the liver, but rather through direct activation of hepatic insulin signaling. Article HighlightsO_LIMorning insulin delivery primes the liver for increased hepatic glucose uptake (HGU) later in the day, but the mechanism (direct hepatic and/or indirect insulin action) remains unclear. C_LIO_LIThis study compared insulin infusion via physiologic (hepatic portal vein) and clinical (peripheral) routes to assess their impact on afternoon hepatic glucose disposal. C_LIO_LIMorning peripheral insulin delivery failed to induce a significant enhancing effect on afternoon HGU and glycogen storage, unlike morning hepatic portal vein insulin delivery, which did. C_LIO_LIThese findings highlight the importance of achieving appropriate hepatic insulin exposure in the morning to effectively prime the liver for efficient glucose disposal. C_LI

Ketone body (KB) utilization increases during fasting and exercise due to enhanced hepatic fatty acid oxidation and KB production via the rate-limiting mitochondrial enzyme hydroxymethylglutaryl-CoA synthase (HMGCS2). Since KB metabolism intersects with multiple metabolic pathways, and skeletal muscle KB catabolism rises during exercise, we tested the hypothesis that liver-specific HMGCS2 knockouts (KO) would have reduced energy expenditure (EE) and changes in the mitochondrial proteome of skeletal muscle with chronic exercise through voluntary wheel running (VWR), time-restricted feeding (TRF), or both combined to boost hepatic KB production and utilization. Control (CON) and HMGCS2 knockout (KO) mice (n=6-8 per group) underwent sedentary ad libitum feeding (SED+AL), SED+TRF, VWR+AL, and VWR+TRF for 16 weeks, with whole-body EE measured using indirect calorimetry. In CON mice, VWR increased total EE by 19.5% and non-resting EE by 50% under AL conditions, and total EE by 16% and non-resting EE by 47.9% under TRF conditions. However, the EE increases seen with VWR did not occur in KO mice. Proteomic analysis revealed that the loss of liver HMGCS2 significantly impacted proteins involved in metabolic processes within skeletal muscle, including reduced oxidative phosphorylation (OXPHOS) protein expression in SED KO mice compared to sedentary CON. Notably, VWR restored OXPHOS protein expression in the muscle of the liver HMGCS2 KO but did not alter it in the CON. Furthermore, muscle from liver HMGCS2 KO mice had elevated expression glycolytic pathways in sedentary and VWR conditions. These results indicate that hepatic ketogenic deficiency (HMGCS2 KO) diminishes exercise-induced increases in EE and uniquely impacts baseline and exercise-related adaptations in the metabolic and mitochondrial proteome of skeletal muscle.

Regulation of lipid metabolism is fundamental for metabolic health, and adipose tissue is a central component in this process. Adipose tissue differs dramatically between women and men with a higher subcutaneous capacity for storage and healthy metabolism in women. Sex hormone-binding globulin (SHBG) contributes to the regulation of circulating sex hormone bioavailability and has been shown to predict risk of metabolic dysfunction. We here investigate the sex-specific relationship of SHBG with metabolic status and adipocyte-dependent lipolysis. We measured serum concentrations of sex hormones, SHBG, fasting glucose and insulin in a cohort of 63 women and 27 men from which adipose biopsies were collected and mature adipocytes were isolated. We found that, in women, high serum SHBG concentrations were strongly associated with low HOMA-IR in vivo, and lower baseline lipolysis but higher responsiveness to isopropanol-induced lipolysis ex vivo. In contrast, no effect of SHBG on the above-mentioned parameters were observed in men. In vitro, cultured adipocytes also increased lipolytic capacity in response to SHBG, but only in the absence of testosterone, suggesting that testosterone inhibits the catecholmine-induced lipolysis of SHBG in adipose tissue. In conclusion, we here define a novel role for SHBG in adipocyte lipolysis. At the same time, our data emphasize sex-dependent differences in adipocyte lipid metabolism, and we propose testosterone binding to SHBG as a driving factor mediating these differences.

Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with body mass index and obesity-related pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with FABP4 deletion in cells known to express the gene; adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was only reduced by [~]25% in Adipo-KO mice, whereas Endo-KO mice showed [~]75% decreases compared to wildtype controls. In contrast, Adipo-KO mice exhibited [~]62% reduction in FABP4 responses to lipolysis, while there was minimal reduction in Endo-KO mice, indicating that adipocytes are the main FABP4 source in lipolysis. We did not detect any myeloid cell contribution to circulating FABP4. Surprisingly, despite the nearly intact FABP4 responses, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/511807v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1b2d566org.highwire.dtl.DTLVardef@1d82083org.highwire.dtl.DTLVardef@3e9726org.highwire.dtl.DTLVardef@1357648_HPS_FORMAT_FIGEXP M_FIG C_FIG

PURPOSEBecause higher adiposity is associated with cardiometabolic disease, we assessed the relationship between body composition (body fat percentage; BF%) and postprandial metabolic flexibility. METHODSYoung adults (n = 27, n = 15 females, BMI = 27.1 {+/-} 4.5; BF% = 30.4 {+/-} 8.7) without overt pathology completed a 100g oral glucose tolerance test (OGTT). Indirect calorimetry before (fasting) and following (30, 60, 90, 120 min) consumption was used to calculate respiratory exchange ratio (RER) and oxidation of carbohydrates (CHO) and fats (FOX). Serum and plasma were collected at corresponding time points and analyzed for blood glucose, insulin, and NEFAs. Data from individuals with normal weight were compared to those with overweight/obesity by two-way repeated measures ANVOA. The effect of BF% on postprandial metabolism was tested via linear mixed models while adjusting for potential confounders. RESULTSDuring the OGTT, blood glucose, serum insulin, plasma lactate, RER, and CHO all significantly increased while plasma NEFAs and whole-body FOX decreased (all p<0.05). BF% modified the relationship between postprandial RER and time (p = 0.019) as well as postprandial CHO and time (p = 0.023) without an effect of BF% on FOX; individuals with higher BF% increase their RER and CHO faster and to a greater extent than those with lower BF%. CONCLUSIONBody fat percentage is associated with greater postprandial metabolic flexibility during an OGTT in young adults. Despite increased adiposity, metabolic flexibility may be preserved, representing a compensatory adaptation to decreased glucose storage in the postprandial period.

To examine factors underlying why most, but not all adults with obesity exhibit impaired insulin-mediated glucose uptake, we compared: 1) rates of fatty acid (FA) release from adipose tissue, 2) skeletal muscle lipid droplet (LD) characteristics, and 3) insulin signaling events in skeletal muscle collected from cohorts of adults with obesity with "HIGH" versus "LOW" insulin sensitivity for glucose uptake. Seventeen adults with obesity (BMI: 36{+/-}3kg/m2) completed a 2h hyperinsulinemic-euglycemic clamp with stable isotope tracer infusions to measure glucose rate of disappearance (glucose Rd) and FA rate of appearance (FA Ra). Skeletal muscle biopsies were collected at baseline and 30min into the insulin infusion. Participants were stratified into HIGH (n=7) and LOW (n=10) insulin sensitivity cohorts by their glucose Rd during the hyperinsulinemic clamp (LOW<400; HIGH>550 nmol/kgFFM/min/[{micro}U/mL]). Insulin-mediated suppression of FA Ra was lower in LOW compared with HIGH (p<0.01). In skeletal muscle, total intramyocellular lipid content did not differ between cohorts. However, the size of LDs in the subsarcolemmal region (SS) of type II muscle fibers was larger in LOW compared with HIGH (p=0.01). Additionally, insulin receptor (IR) interactions with regulatory proteins CD36 and Fyn were lower in LOW versus HIGH (p<0.01), which aligned with attenuated insulin-mediated Tyr phosphorylation of IR{beta} and downstream insulin-signaling proteins in LOW. Collectively, reduced ability for insulin to suppress FA mobilization, with accompanying modifications in intramyocellular LD size and distribution, and diminished IR interaction with key regulatory proteins may be key contributors to impaired insulin-mediated glucose uptake commonly found in adults with obesity. KEY POINTSO_LIAlthough most adults with obesity exhibit impaired insulin-mediated glucose uptake (insulin resistance), some remain sensitive to insulin. Factors that "protect" adults with obesity from developing resistance to insulin-mediated glucose uptake are poorly understood. C_LIO_LIPotent suppression of fatty acid (FA) mobilization from adipose tissue by insulin is a strong predictor of whole-body insulin-mediated glucose uptake. C_LIO_LIParticipants with higher sensitivity for insulin-mediated glucose uptake had smaller intramyocellular lipid droplets (LDs) within the subsarcolemmal region of type II skeletal muscle fibers. C_LIO_LINovel findings revealed that insulin receptor (IR) interaction with the long-chain fatty acid transport protein, CD36, and the Src-family kinase, Fyn, directly associated with higher rates of glucose uptake under basal and hyperinsulinemic conditions. C_LIO_LITogether, the findings suggest impaired suppression of FA release from adipose tissue associates with reduced glucose uptake in skeletal muscle due in part to a defect in IR activation by CD36/Fyn and altered subcellular LD characteristics. C_LI

IntroductionGCN5 (Kat2a) is a lysine acetyl transferase capable of acetylating and inhibiting PGC-1 activity. As such, it is described as a negative regulator of PGC-1 and subsequently restricts mitochondrial content. However, elimination of GCN5 in skeletal muscle does not increase mitochondrial content or alter lipid metabolism under normal metabolic conditions. GCN5 levels increase with high-fat diet (HFD) feeding in rodents. Additionally, the GCN5 homolog, PCAF, has previously been shown to also acetylate and inhibit PGC-1 and therefore may possibly compensate for loss of GCN5. ObjectiveThe objective of this study was to examine if with HFD feeding that elimination of GCN5 (Kat2a gene) from skeletal muscle would elicit improvements in mitochondrial and metabolic markers. MethodsSkeletal muscle specific GCN5 knockouts (Gcn5 skm-/-) were fed an HFD. Body composition, cardio-metabolic and physical fitness outcomes were monitored. Additionally, cultured myotubes were treated with a pan-GCN5/PCAF inhibitor and examined for changes in mitochondrial markers. ResultsElimination of skeletal muscle GCN5 did not alter body composition, tissue masses, energy intake, or energy expenditure measurements of mice fed an HFD. Furthermore, whole body glucose homeostasis and cardiac measurements were not altered. There were few differences in lipid metabolism genes, relatively more glucose oxidation versus Gcn5 skm+/+ (wildtype) mice, and a reduction in Pdk4 expression. Exercise capacity and mitochondrial content levels were not altered in Gcn5 skm-/- mice. Further, elimination of GCN5 in skeletal muscle increased Kat2b (PCAF) mRNA expression; however, inhibition of GCN5/PCAF bromodomains in cultured myotubes did not increase oxidative metabolism genes and decreased expression of some mitochondrial genes and Pdk4 mRNA. ConclusionsNeither elimination of GCN5, nor simultaneous inhibition of GCN5 and its homolog PCAF improved skeletal muscle mitochondrial content under normal or HFD-fed conditions. Despite this, GCN5 may play a role in regulating macronutrient preference by regulating Pdk4 content. Thus, HFD/macronutrient excess revealed novel roles of GCN5 in skeletal muscle. Highlights- Skeletal muscle specific elimination of Gcn5/Kat2a decreases fat oxidation without 1) preventing high-fat diet induced weight gain, 2) improving whole body glucose handling, or 3) improving skeletal muscle mitochondrial content. - Inhibition of the GCN5 and PCAF bromodomains and Gcn5 ablation decreases expression of Pdk4. - Expression of Kat2b increases with Gcn5 elimination in skeletal muscle. - Inhibition of the GCN5 and PCAF bromodomains do not result in increased skeletal muscle mitochondrial content.

Background and aimsHepatic insulin action is essential for whole body glucose homeostasis. Insulins inhibition of glycogen breakdown, suppression of gluconeogenesis, and activation of glycogen synthesis are critical for postprandial glucose disposal. AKT, a serine-threonine kinase and well-established insulin signaling target, regulates hepatic glucose metabolism through transcriptional and posttranslational mechanisms. However, current knowledge about AKTs regulation of hepatic glucose metabolism largely stems from genetic loss of function models, precluding observation of rapid, transcription-independent effects. MethodsStable isotope tracing using [U-13C]-glucose was coupled with pharmacological inhibition of AKT using MK-2206 in primary rat hepatocytes. Bulk metabolomics was performed on AKT knockout livers and primary rat hepatocytes treated with MK-2206. Radiolabeled glucose was used to quantify short-term changes to glycogen synthesis. ResultsMK-2206 treatment decreased glucose contribution to glucose 6-phosphate and uridine diphosphate glucose within minutes without significantly affecting total metabolite pool sizes or changes to glucokinase protein levels. This was accompanied by a decrease in glucose contribution to glycogen, independent of changes to glycogen breakdown or glycogen synthase phosphorylation. ConclusionsTogether, these results demonstrate that AKT acutely regulates glucose contribution to glycogen and its upstream precursors, suggesting a transcription-independent, glucokinase-centered mechanism for glycogen synthesis through the direct pathway.

The Staub-Traugott effect describes the improved glycemic response observed after consuming a second identical meal. We previously showed that morning (AM) hyperinsulinemia primes the liver for enhanced afternoon (PM) meal-associated net hepatic glucose uptake (NHGU) and glycogen storage. However, depending on the meal composition, both insulin and glucagon may rise. Therefore, we investigated whether AM hyperglucagonemia alters the priming effect of AM hyperinsulinemia on subsequent hepatic glucose metabolism. Dogs underwent a 4h AM hyperinsulinemic-euglycemic clamp paired with either basal (AM INS, n=8) or elevated glucagon (AM INS+GCG, n=8). After a 1.5h rest, dogs underwent a 2.5h PM hyperinsulinemic-hyperglycemic clamp designed to mimic postprandial conditions. AM hyperglucagonemia reduced PM NHGU through additive shifts in both hepatic glucose uptake and production, leading to lower direct glycogen synthesis and less glycolytic flux. Mechanistically, hepatic glucokinase protein was reduced in the AM INS+GCG vs. AM INS group, suggesting diminished capacity for glucose phosphorylation and lower intracellular G6P, a central node regulating downstream hepatic glucose metabolism. Thus, morning hepatic glucagon exposure diminishes insulins ability to prime the liver, limiting net hepatic glucose uptake during a later meal. These results underscore how antecedent hormonal signals govern subsequent postprandial hepatic glucose metabolism.

ObjectiveWhite adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. WAT absence or loss occurring through lipodystrophy and lipoatrophy contributes to the development of dyslipidemia, hepatic steatosis, and insulin resistance. We previously demonstrated the sole small ubiquitin-like modifier (SUMO) E2-conjuguating enzyme Ubc9 represses human adipocyte differentiation. Germline and other tissue-specific deletions of Ubc9 frequently cause lethality in mice. As a result, the role of Ubc9 during WAT development remains unknown. MethodsTo determine how Ubc9 impacts body composition and energy balance, we generated adipocyte-specific Ubc9 knockout mice (Ubc9a-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ubc9 locus. Subsequent genetic crosses to AdipoQ-Cre transgenic mice allowed deletion of Ubc9 in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ubc9a-KO and littermate controls during postnatal growth. ResultsTo our surprise, Ubc9a-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period. ConclusionsOur results demonstrate that Ubc9 expression in mature adipocytes is essential for maintaining WAT expansion. Deletion of Ubc9 in fat cells compromised and diminished adipocyte function that provoked WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ubc9 during white adipocyte expansion and endocrine control of energy balance.

Aims/hypothesisTo investigate if there is a causal relationship between changes in insulin resistance and mitochondrial respiratory function and content in rats fed a high fat diet (HFD) with or without concurrent exercise training. We hypothesised that provision of a high fat diet (HFD) would increase insulin resistance and decrease mitochondrial characteristics (content and function), and that exercise training would improve both mitochondrial characteristics and insulin resistance in rats fed a HFD. MethodsMale Wistar rats were given either a chow diet or a high fat diet (HFD) for 12 weeks. After 4 weeks of the dietary intervention, half of the rats in each group began eight weeks of interval training. In vivo glucose and insulin tolerance was assessed, as was ex vivo glucose uptake in epitrochlearis muscle. Mitochondrial respiratory function was assessed in permeabilised soleus and white gastrocnemius (WG) muscles. Mitochondrial content was determined by measurement of citrate synthase (CS) activity and protein expression of components of the electron transport system (ETS). ResultsHFD rats had impaired glucose and insulin tolerance. HFD did not change CS activity in the soleus; however, it did increase CS activity in WG (Chow 5.9 {+/-} 0.5, HFD 7.2 {+/-} 0.7 mol h-1 kg protein-1). Protein expression of components of the ETS and mitochondrial respiratory function (WG Chow 65.2 {+/-} 8.4, HFD 88.6 {+/-} 8.7 pmol O2 s-1 mg-1) were also increased by HFD. Exercise training improved glucose and insulin tolerance in the HFD rats. Exercise training did not alter CS activity in either muscle. Mitochondrial respiratory function was increased with exercise training in the chow fed animals in soleus muscle, but not in WG. This exercise effect was absent in the HFD animals. Mitochondrial characteristics did not consistently correlate with insulin or glucose tolerance. Conclusions/interpretationHFD induced insulin resistance, but it did not negatively affect any of the measured mitochondrial characteristics. Exercise training improved insulin resistance, but without changes in mitochondrial respiration and content. The lack of an association between mitochondrial characteristics and insulin resistance was reinforced by the absence of strong correlations between these measures. Our results suggest that defects in mitochondrial respiration and content are not responsible for insulin resistance in HFD rats.

Cellular heterogeneity of human adipose tissue, is linked to the pathophysiology of obesity and may impact the response to energy restriction and changes in fat mass. Here, we provide an optimized pipeline to estimate cellular composition in human abdominal subcutaneous adipose tissue (ASAT) from publicly available bulk RNA-Seq using signature profiles from our previously published full-length single nuclei (sn)RNA-Seq of the same depot. Individuals with obesity had greater proportions of macrophages and lower proportions of adipocyte sub-populations and vascular cells compared with lean individuals. Two months of diet-induced weight loss (DIWL) increased the estimated proportions of macrophages; however, two years of DIWL reduced the estimated proportions of macrophages, thereby suggesting a bi-phasic nature of cellular remodeling of ASAT during weight loss. Our optimized high-throughput pipeline facilitates the assessment of composition changes of highly characterized cell types in large numbers of ASAT samples using low-cost bulk RNA-Seq. Our data reveal novel changes in cellular heterogeneity and its association with cardiometabolic health in humans with obesity and following weight loss. Lead contactKatie Whytock (Katie.Whytock@adventhealth.com)

Fatty acid binding protein 4 (FABP4) is linked with the pathogenesis of metabolic diseases, including diabetes and cardiovascular disease in both mice and humans. It has also been demonstrated that the levels of hormonal FABP4 are strongly associated with obesity, and secretion is stimulated under conditions of fasting and lipolysis both in vivo and in vitro. Here, we utilized adipocyte-specific deficiency of adipose triglyceride lipase (ATGL) in a mouse model (ATGLAdpKO) to evaluate the regulation of FABP4 secretion by lipolytic signals in the absence of actual lipolysis in vivo. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of ATGL, and from adipose tissue explants from ATGLAdpKO mice. Unexpectedly, upon activation beta-adrenergic receptors, ATGLAdpKO mice exhibited significantly higher levels of circulating FABP4 as compared to ATGLfl/fl controls in vivo, with no corresponding increase in non-esterified free fatty acids or glycerol, confirming the lack of lipolysis. We also generated an additional model with adipocyte-specific deletion of FABP4 in the background of ATGLAdpKO mice (ATGL/FABP4AdpKO or DKO) to evaluate the cellular source of circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the elevated FABP4 hormone levels in the ATGLAdpKO mice were indeed from the adipocytes. ATGLAdpKO mice did not exhibit an increase in insulin secretion upon stimulation of lipolysis, but had a normal insulin response to glucose injection along with increased FABP4 secretion, suggesting the elevated FABP4 secretion is not due to lack of insulin. Inhibition of sympathetic signaling during lipolysis using hexamethonium significantly reduced FABP4 secretion in ATGLAdpKO mice compared to controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for stimulated in vivo FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.

Background & AimsThe hepatic glucagon-PKA-CREB signaling axis plays a central role in regulating gluconeogenesis and maintaining glucose homeostasis during fasting. However, the mechanisms that govern the spatial coordination and substrate specificity of this pathway remain incompletely understood. This study determines the role of the scaffolding protein RACK1 (Receptor for Activated C Kinase 1) in orchestrating glucagon signaling to regulate hepatic gluconeogenesis. MethodsRACK1 was acutely deleted in mouse liver and primary hepatocytes. Metabolic phenotypes were assessed by glucose, pyruvate, and insulin tolerance tests, and hepatocyte glucose production assays. Protein interactions were analyzed with co-immunoprecipitation, GST pulldown, and proximity ligation assays. Subcellular localization and signaling events were studied by Western blot, confocal microscopy, and fractionation. Functional rescue was performed by hepatic expression of a constitutively active PKA catalytic subunit (PKAcW196R). ResultsAcute hepatic RACK1 deficiency caused fasting hypoglycemia, impaired gluconeogenesis, and improved glucose and pyruvate tolerance without affecting insulin signaling. RACK1 directly bound GCGR, PKA regulatory (RII) and catalytic (PKAc) subunits, and CREB, functioning as a dual-compartment scaffold assembling GCGR-PKA complexes at the plasma membrane and PKAc-CREB complexes in the nucleus. Loss of RACK1 impaired PKAc translocation, CREB phosphorylation, and gluconeogenic gene expression. These defects were rescued by PKAcW196R expresson. Overexpression of RACK1 WD1-2 and WD3-4 domains, which mediate PKA and GCGR interactions, similarly disrupted PKA signaling and gluconeogenesis. ConclusionRACK1 spatially organizes the glucagon-PKA-CREB axis, ensuring precise signal propagation and efficient hepatic gluconeogenesis, revealing a novel mechanism of compartmentalized signal regulation in glucose metabolism.

Insulin-stimulated glucose uptake (ISGU) in adipocytes is central to maintain systemic glucose homeostasis. Understanding how ISGU relates to adipocyte traits, such as cell-size, is critical for elucidating pressing questions related to metabolic dysfunction connected to adipose hypertrophy and hyperplasia. Cell size is considered a central trait reflecting multiple aspects of adipocyte metabolism. However, a robust quantitative approach to estimate ISGU for a specific cell size is currently missing. Here, in an attempt to move towards such a method, we have formulated an approach using a mathematical framework. The framework consists of a linear equation: the product of the known number of cells (calculated using coulter counter-based cell-size distributions) and the unknown ISGU/cell, compared to the absolute ISGU (measured using 14C-glucose tracer assays). To solve this equation, we formulate a minimization problem which is optimized to find the unknown ISGU/cell for the best solution. Using different formulations of the equation we can investigate the need for either cell size-dependent or independent ISGU/cell, to describe varying glucose uptake in a cell sample of various cell sizes. While the framework needs further refinement, we demonstrate that cell size-dependent uptake slightly improved the agreement between model and experimental data for some groups. Together, with further validation this could serve as a useful tool to resolve long-standing questions regarding size-dependent characteristics like adipocyte size and cellular function. Key findingsHerein we explore a method to quantify cell size-dependent glucose uptake in adipocytes

BackgroundExposure to high maternal adiposity in utero is a significant risk factor for the later-life development of metabolic syndrome (MetS), including non-alcoholic fatty liver disease (NAFLD). We have previously shown that high pre-pregnancy adiposity programs adipose tissue dysfunction in the offspring, leading to spillover of fatty acids into the circulation, a key pathogenic event in obesity-associated MetS. Herein, we hypothesized that programming of adipose tissue dysfunction in offspring born to overweight dams increases the risk for developing NAFLD. ResultsFemales heterozygous for leptin receptor deficiency (Hetdb) were used as a model of high pre-pregnancy adiposity. Wild-type (Wt) offspring born to Hetdb pregnancies gained significantly more body fat following high fat/fructose diet (HFFD) compared to Wt offspring born to Wt dams. HFFD increased circulating free fatty acids (FFA) in male offspring of control dams, while FFA levels were similar in HFFD-fed offspring from Wt dams compared to CD or HFFD-Wt offspring from Hetdb dams. Despite female-specific protection from diet-induced FFA spillover, both male and female offspring from Hetdb. dams were more susceptible to diet-induced hepatosteatosis. Lipidomic analysis revealed that CD-offspring of overweight dams had decreased hepatic PUFA levels compared to control offspring. Changes to saturated fatty acids (SFA) and the de novo lipogenic (DNL) index were diet driven; however, there was a significant effect of the intrauterine environment on FA elongation and {Delta}9 desaturase activity. ConclusionHigh maternal adiposity during pregnancy programs a susceptibility to diet-induced hepatosteatosis.

Transcription factor 7-like 2 (TCF7L2) is a downstream effector of the Wnt/beta-catenin signalling pathway and its expression is critical for adipocyte development. The precise role of TCF7L2 in glucose and lipid metabolism in adult adipocytes remains to be defined. Here, we aim to investigate how changes in TCF7L2 expression in mature adipocytes affect glucose homeostasis. Tcf7l2 was selectively ablated from mature adipocytes in C57BL/6J mice using an adiponectin promoter-driven Cre recombinase to recombine alleles floxed at exon 1 of the Tcf7l2 gene. Mice lacking Tcf7l2 in mature adipocytes displayed normal body weight. Male mice exhibited normal glucose homeostasis at eight weeks of age. Male heterozygote knockout mice (aTCF7L2het) exhibited impaired glucose tolerance (AUC increased 1.14 {+/-} 0.04 -fold, p=0.03), as assessed by intraperitoneal glucose tolerance test, and changes in fat mass at 16 weeks (increased by 1.4 {+/-} 0.09-fold, p=0.007). Homozygote knockout mice exhibited impaired oral glucose tolerance at 16 weeks of age (AUC increased 2.15 {+/-} 0.15-fold, p=0.0001). Islets of Langerhans exhibited impaired glucose-stimulated insulin secretion in vitro (decreased 0.54 {+/-} 0.13-fold aTCF7L2KO vs control, p=0.02), but no changes in in vivo glucose-stimulated insulin secretion. Female mice in which one or two alleles of the Tcf7l2 gene was knocked out in adipocytes displayed no changes in glucose tolerance, insulin sensitivity or insulin secretion. Plasma levels of glucagon-like peptide-1 and gastric inhibitory polypeptide were lowered in knockout mice (decreased 0.57 {+/-} 0.03-fold and 0.41 {+/-} 0.12-fold, p=0.04 and p=0.002, respectively), whilst plasma free fatty acids and Fatty Acid Binding Protein 4 circulating levels were increased by 1.27 {+/-} 0.07 and 1.78 {+/-} 0.32-fold, respectively (p=0.05 and p=0.03). Mice with biallelic Tcf7l2 deletion exposed to high fat diet for 9 weeks exhibited impaired glucose tolerance (p=0.003 at 15 min after glucose injection) which was associated with reduced in vivo glucose-stimulated insulin secretion (decreased 0.51 {+/-} 0.03-fold, p=0.02). Thus, our data indicate that loss of Tcf7l2 gene expression in adipocytes leads to impairments on metabolic responses which are dependent on gender, age and nutritional status. Our findings further illuminate the role of TCF7L2 in the maintenance of glucose homeostasis.