Of mice and men: Pinpointing species differences in adipose tissue biology.

The prevalence of obesity and metabolic diseases continues to rise, which has led to an increased interest in studying adipose tissue to elucidate underlying disease mechanisms. The use of genetic mouse models has been critical for understanding the role of specific genes for adipose tissue function and the tissue's impact on other organs. However, mouse adipose tissue displays key differences to human fat, which has led, in some cases, to the emergence of some confounding concepts in the adipose field. Such differences include the depot-specific characteristics of visceral and subcutaneous fat, and divergences in thermogenic fat phenotype between the species. Adipose tissue characteristics may therefore not always be directly compared between species, which is important to consider when setting up new studies or interpreting results. This mini review outlines our current knowledge about the cell biological differences between human and mouse adipocytes and fat depots, highlighting some examples where inadequate knowledge of species-specific differences can lead to confounding results, and presenting plausible anatomic explanations that may underlie the differences. The article thus provides critical insights and guidance for researchers working primarily with only human or mouse fat tissue, and may contribute to new ideas or concepts in the important and evolving field of adipose biology.

Chronotherapy with a glucokinase activator profoundly improves metabolism in obese Zucker rats.

Circadian rhythms play a critical role in regulating metabolism, including daily cycles of feeding/fasting. Glucokinase (GCK) is central for whole-body glucose homeostasis and oscillates according to a circadian clock. GCK activators (GKAs) effectively reduce hyperglycemia, but their use is also associated with hypoglycemia, hyperlipidemia, and hepatic steatosis. Given the circadian rhythmicity and natural postprandial activation of GCK, we hypothesized that GKA treatment would benefit from being timed specifically during feeding periods. Acute treatment of obese Zucker rats with the GKA AZD1656 robustly increased flux into all major metabolic pathways of glucose disposal, enhancing glucose elimination. Four weeks of continuous AZD1656 treatment of obese Zucker rats improved glycemic control; however, hepatic steatosis and inflammation manifested. In contrast, timing AZD1656 to feeding periods robustly reduced hepatic steatosis and inflammation in addition to improving glycemia, whereas treatment timed to fasting periods caused overall detrimental metabolic effects. Mechanistically, timing AZD1656 to feeding periods diverted newly synthesized lipid toward direct VLDL secretion rather than intrahepatic storage. In line with increased hepatic insulin signaling, timing AZD1656 to feeding resulted in robust activation of AKT, mTOR, and SREBP-1C after glucose loading, pathways known to regulate VLDL secretion and hepatic de novo lipogenesis. In conclusion, intermittent AZD1656 treatment timed to feeding periods promotes glucose disposal when needed the most, restores metabolic flexibility and hepatic insulin sensitivity, and thereby avoids hepatic steatosis. Thus, chronotherapeutic approaches may benefit the development of GKAs and other drugs acting on metabolic targets.

Cellular senescence in hepatocytes contributes to metabolic disturbances in NASH.

Cellular senescence is a state of irreversible cell cycle arrest and has been shown to play a key role in many diseases, including metabolic diseases. To investigate the potential contribution of hepatocyte cellular senescence to the metabolic derangements associated with non-alcoholic steatohepatitis (NASH), we treated human hepatocyte cell lines HepG2 and IHH with the senescence-inducing drugs nutlin-3a, doxorubicin and etoposide. The senescence-associated markers p16, p21, p53 and beta galactosidase were induced upon drug treatment, and this was associated with increased lipid storage, increased expression of lipid transporters and the development of hepatic steatosis. Drug-induced senescence also led to increased glycogen content, and increased VLDL secretion from hepatocytes. Senescence was also associated with an increase in glucose and fatty acid oxidation capacity, while de novo lipogenesis was decreased. Surprisingly, cellular senescence caused an overall increase in insulin signaling in hepatocytes, with increased insulin-stimulated phosphorylation of IR, Akt, and MAPK. Together, these data indicate that hepatic senescence plays a causal role in the development of NASH pathogenesis, by modulating glucose and lipid metabolism, favoring steatosis. Our findings contribute to a better understanding of the mechanisms linking cellular senescence and fatty liver disease and support the development of new therapies targeting senescent cells for the treatment of NASH.

BMP4 and Gremlin 1 regulate hepatic cell senescence during clinical progression of NAFLD/NASH.

The role of hepatic cell senescence in human non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is not well understood. To examine this, we performed liver biopsies and extensive characterization of 58 individuals with or without NAFLD/NASH. Here, we show that hepatic cell senescence is strongly related to NAFLD/NASH severity, and machine learning analysis identified senescence markers, the BMP4 inhibitor Gremlin 1 in liver and visceral fat, and the amount of visceral adipose tissue as strong predictors. Studies in liver cell spheroids made from human stellate and hepatocyte cells show BMP4 to be anti-senescent, anti-steatotic, anti-inflammatory and anti-fibrotic, whereas Gremlin 1, which is particularly highly expressed in visceral fat in humans, is pro-senescent and antagonistic to BMP4. Both senescence and anti-senescence factors target the YAP/TAZ pathway, making this a likely regulator of senescence and its effects. We conclude that senescence is an important driver of human NAFLD/NASH and that BMP4 and Gremlin 1 are novel therapeutic targets.

Chronic hyperinsulinemia promotes human hepatocyte senescence.

OBJECTIVE: Cellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. METHODS: Using fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. RESULTS: Results showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. CONCLUSION: Our findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

Lipoxins reduce obesity-induced adipose tissue inflammation in 3D-cultured human adipocytes and explant cultures.

Adipose tissue inflammation drives obesity-related cardiometabolic diseases. Enhancing endogenous resolution mechanisms through administration of lipoxin A4, a specialized pro-resolving lipid mediator, was shown to reduce adipose inflammation and subsequently protects against obesity-induced systemic disease in mice. Here, we demonstrate that lipoxins reduce inflammation in 3D-cultured human adipocytes and adipose tissue explants from obese patients. Approximately 50% of patients responded particularly well to lipoxins by reducing inflammatory cytokines and promoting an anti-inflammatory M2 macrophage phenotype. Responding patients were characterized by elevated systemic levels of C-reactive protein, which causes inflammation in cultured human adipocytes. Responders appeared more prone to producing anti-inflammatory oxylipins and displayed elevated prostaglandin D2 levels, which has been interlinked with transcription of lipoxin-generating enzymes. Using explant cultures, this study provides the first proof-of-concept evidence supporting the therapeutic potential of lipoxins in reducing human adipose tissue inflammation. Our data further indicate that lipoxin treatment may require a tailored personalized-medicine approach.

The SGLT2 inhibitor dapagliflozin promotes systemic FFA mobilization, enhances hepatic ß-oxidation, and induces ketosis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-3H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (Ra), whole-body FFA oxidation (Rox), and nonoxidative disposal (Rst). In the liver, clearance (Kß-ox) and flux (Rß-ox) of FFA into ß-oxidation were estimated using [9,10-3H]-(R)-bromopalmitate/[U-14C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, Ra, Rox, and Rst with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to ß-oxidation, with increased Kß-ox, Rß-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma ß-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward ß-oxidation.

Obesity and hyperinsulinemia drive adipocytes to activate a cell cycle program and senesce.

Obesity is considered an important factor for many chronic diseases, including diabetes, cardiovascular disease and cancer. The expansion of adipose tissue in obesity is due to an increase in both adipocyte progenitor differentiation and mature adipocyte cell size. Adipocytes, however, are thought to be unable to divide or enter the cell cycle. We demonstrate that mature human adipocytes unexpectedly display a gene and protein signature indicative of an active cell cycle program. Adipocyte cell cycle progression associates with obesity and hyperinsulinemia, with a concomitant increase in cell size, nuclear size and nuclear DNA content. Chronic hyperinsulinemia in vitro or in humans, however, is associated with subsequent cell cycle exit, leading to a premature senescent transcriptomic and secretory profile in adipocytes. Premature senescence is rapidly becoming recognized as an important mediator of stress-induced tissue dysfunction. By demonstrating that adipocytes can activate a cell cycle program, we define a mechanism whereby mature human adipocytes senesce. We further show that by targeting the adipocyte cell cycle program using metformin, it is possible to influence adipocyte senescence and obesity-associated adipose tissue inflammation.

A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes.

Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro. The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021).

Adult mice are unresponsive to AAV8-Gremlin1 gene therapy targeting the liver.

OBJECTIVE: Gremlin 1 (GREM1) is a secreted BMP2/4 inhibitor which regulates commitment and differentiation of human adipose precursor cells and prevents the browning effect of BMP4. GREM1 is an insulin antagonist and serum levels are high in type 2 diabetes (T2D). We here examined in vivo effects of AAV8 (Adeno-Associated Viral vectors of serotype eight) GREM 1 targeting the liver in mature mice to increase its systemic secretion and also, in a separate study, injected recombinant GREM 1 intraperitoneally. The objective was to characterize systemic effects of GREM 1 on insulin sensitivity, glucose tolerance, body weight, adipose cell browning and other local tissue effects. METHODS: Adult mice were injected with AAV8 vectors expressing GREM1 in the liver or receiving regular intra-peritoneal injections of recombinant GREM1 protein. The mice were fed with a low fat or high fat diet (HFD) and followed over time. RESULTS: Liver-targeted AAV8-GREM1 did not alter body weight, whole-body glucose and insulin tolerance, or adipose tissue gene expression. Although GREM1 protein accumulated in liver cells, GREM1 serum levels were not increased suggesting that it may not have been normally processed for secretion. Hepatic lipid accumulation, inflammation and fibrosis were also not changed. Repeated intraperitoneal rec-GREM1 injections for 5 weeks were also without effects on body weight and insulin sensitivity. UCP1 was slightly but significantly reduced in both white and brown adipose tissue but this was not of sufficient magnitude to alter body weight. We validated that recombinant GREM1 inhibited BMP4-induced pSMAD1/5/9 in murine cells in vitro, but saw no direct inhibitory effect on insulin signalling and pAkt (ser 473 and thr 308) activation. CONCLUSION: GREM1 accumulates intracellularly when overexpressed in the liver cells of mature mice and is apparently not normally processed/secreted. However, also repeated intraperitoneal injections were without effects on body weight and insulin sensitivity and adipose tissue UCP1 levels were only marginally reduced. These results suggest that mature mice do not readily respond to GREMLIN 1 but treatment of murine cells with GREMLIN 1 protein in vitro validated its inhibitory effect on BMP4 signalling while insulin signalling was not altered.

Lipid and glucose metabolism in white adipocytes: pathways, dysfunction and therapeutics.

In mammals, the white adipocyte is a cell type that is specialized for storage of energy (in the form of triacylglycerols) and for energy mobilization (as fatty acids). White adipocyte metabolism confers an essential role to adipose tissue in whole-body homeostasis. Dysfunction in white adipocyte metabolism is a cardinal event in the development of insulin resistance and associated disorders. This Review focuses on our current understanding of lipid and glucose metabolic pathways in the white adipocyte. We survey recent advances in humans on the importance of adipocyte hypertrophy and on the in vivo turnover of adipocytes and stored lipids. At the molecular level, the identification of novel regulators and of the interplay between metabolic pathways explains the fine-tuning between the anabolic and catabolic fates of fatty acids and glucose in different physiological states. We also examine the metabolic alterations involved in the genesis of obesity-associated metabolic disorders, lipodystrophic states, cancers and cancer-associated cachexia. New challenges include defining the heterogeneity of white adipocytes in different anatomical locations throughout the lifespan and investigating the importance of rhythmic processes. Targeting white fat metabolism offers opportunities for improved patient stratification and a wide, yet unexploited, range of therapeutic opportunities.

A Method to Induce Brown/Beige Adipocyte Differentiation from Murine Preadipocytes.

Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.

The colorful versatility of adipocytes: white-to-brown transdifferentiation and its therapeutic potential in humans.

Brown and brite adipocytes contribute to energy expenditure through nonshivering thermogenesis. Though these cell types are thought to arise primarily from the de novo differentiation of precursor cells, their abundance is also controlled through the transdifferentiation of mature white adipocytes. Here, we review recent advances in our understanding of the regulation of white-to-brown transdifferentiation, as well as the conversion of brown and brite adipocytes to dormant, white-like fat cells. Converting mature white adipocytes into brite cells or reactivating dormant brown and brite adipocytes has emerged as a strategy to ameliorate human metabolic disorders. We analyze the evidence of learning from mice and how they translate to humans to ultimately scrutinize the relevance of this concept. Moreover, we estimate that converting a small percentage of existing white fat mass in obese subjects into active brite adipocytes could be sufficient to achieve meaningful benefits in metabolism. In conclusion, novel browning agents have to be identified before adipocyte transdifferentiation can be realized as a safe and efficacious therapy.

Enoxacin induces oxidative metabolism and mitigates obesity by regulating adipose tissue miRNA expression.

MicroRNAs (miRNAs) have been implicated in oxidative metabolism and brown/beige adipocyte identity. Here, we tested whether widespread changes in miRNA expression promoted by treatment with the small-molecule enoxacin cause browning and prevent obesity. Enoxacin mitigated diet-induced obesity in mice, and this was associated with increased energy expenditure. Consistently, subcutaneous white and brown adipose tissues and skeletal muscle of enoxacin-treated mice had higher levels of markers associated with thermogenesis and oxidative metabolism. These effects were cell autonomous since they were recapitulated in vitro in murine and human cell models. In preadipocytes, enoxacin led to a reduction of miR-34a-5p expression and up-regulation of its target genes (e.g., Fgfr1, Klb, and Sirt1), thus increasing FGF21 signaling and promoting beige adipogenesis. Our data demonstrate that enoxacin counteracts obesity by promoting thermogenic signaling and inducing oxidative metabolism in adipose tissue and skeletal muscle in a mechanism that involves, at least in part, miRNA-mediated regulation.

An Obesogenic Dietary Mouse Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD), characterized by steatosis (fat within the liver), inflammation, and fibrosis, which may progress to cirrhosis and hepatocellular carcinoma. Despite the high prevalence, there are currently no approved NASH drug treatments, which urges a faster development of new therapies to address this high unmet medical need. Drug development is facilitated by having reliable and translatable preclinical NASH models. Obesogenic dietary models recapitulate better the natural progression of NASH, with overnutrition and sedentary lifestyle being the main causes. Here we describe the use of a modified version of a diet-induced NASH model, known as the Amylin NASH diet model (AMLN-diet), particularly in the leptin-deficient Lepob/Lepob (ob/ob) mice.

PPARγ and PPARα synergize to induce robust browning of white fat in vivo.

OBJECTIVE: Peroxisome proliferator-activated receptors (PPARs) are key transcription factors that regulate adipose development and function, and the conversion of white into brown-like adipocytes. Here we investigated whether PPARα and PPARγ activation synergize to induce the browning of white fat. METHODS: A selection of PPAR activators was tested for their ability to induce the browning of both mouse and human white adipocytes in vitro, and in vivo in lean and obese mice. RESULTS: All dual PPARα/γ activators tested robustly increased uncoupling protein 1 (Ucp1) expression in both mouse and human adipocytes in vitro, with tesaglitazar leading to the largest Ucp1 induction. Importantly, dual PPARα/γ activator tesaglitazar strongly induced browning of white fat in vivo in both lean and obese male mice at thermoneutrality, greatly exceeding the increase in Ucp1 observed with the selective PPARγ activator rosiglitazone. While selective PPARγ activation was sufficient for the conversion of white into brown-like adipocytes in vitro, dual PPARα/γ activation was superior to selective PPARγ activation at inducing white fat browning in vivo. Mechanistically, the superiority of dual PPARα/γ activators is mediated at least in part via a PPARα-driven increase in fibroblast growth factor 21 (FGF21). Combined treatment with rosiglitazone and FGF21 resulted in a synergistic increase in Ucp1 mRNA levels both in vitro and in vivo. Tesaglitazar-induced browning was associated with increased energy expenditure, enhanced insulin sensitivity, reduced liver steatosis, and an overall improved metabolic profile compared to rosiglitazone and vehicle control groups. CONCLUSIONS: PPARγ and PPARα synergize to induce robust browning of white fat in vivo, via PPARγ activation in adipose, and PPARα-mediated increase in FGF21.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC).

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

BMP4 gene therapy enhances insulin sensitivity but not adipose tissue browning in obese mice.

OBJECTIVE: Bone morphogenetic protein 4 (BMP4) adeno-associated viral vectors of serotype 8 (AAV8) gene therapy targeting the liver prevents the development of obesity in initially lean mice by browning the large subcutaneous white adipose tissue (WAT) and enhancing energy expenditure. Here, we examine whether this approach could also reduce established obesity. METHODS: Dietary-induced obese C57BL6/N mice received AAV8 BMP4 gene therapy at 17-18 weeks of age. They were kept on a high-fat diet and phenotypically characterized for an additional 10-12 weeks. Following termination, the mice underwent additional characterization in vitro. RESULTS: Surprisingly, we observed no effect on body weight, browning of WAT, or energy expenditure in these obese mice, but whole-body insulin sensitivity and glucose tolerance were robustly improved. Insulin signaling and insulin-stimulated glucose uptake were increased in both adipose cells and skeletal muscle. BMP4 also decreased hepatic glucose production and reduced gluconeogenic enzymes in the liver, but not in the kidney, in addition to enhancing insulin action in the liver. CONCLUSIONS: Our findings show that BMP4 prevents, but does not reverse, established obesity in adult mice, while it improves insulin sensitivity independent of weight reduction. The BMP antagonist Noggin was increased in WAT in obesity, which may account for the lack of browning.

Fructose and hepatic insulin resistance.

Excessive caloric intake in a form of high-fat diet (HFD) was long thought to be the major risk factor for development of obesity and its complications, such as fatty liver disease and insulin resistance. Recently, there has been a paradigm shift and more attention is attributed to the effects of sugar-sweetened beverages (SSBs) as one of the culprits of the obesity epidemic. In this review, we present the data invoking fructose intake with development of hepatic insulin resistance in human studies and discuss the pathways by which fructose impairs hepatic insulin action in experimental animal models. First, we described well-characterized pathways by which fructose metabolism indirectly leads to hepatic insulin resistance. These include unequivocal effects of fructose to promote de novo lipogenesis (DNL), impair fatty acid oxidation (FAO), induce endoplasmic reticulum (ER) stress and trigger hepatic inflammation. Additionally, we entertained the hypothesis that fructose can directly impede insulin signaling in the liver. This appears to be mediated by reduced insulin receptor and insulin receptor substrate 2 (IRS2) expression, increased protein-tyrosine phosphatase 1B (PTP1b) activity, whereas knockdown of ketohexokinase (KHK), the rate-limiting enzyme of fructose metabolism, increased insulin sensitivity. In summary, dietary fructose intake strongly promotes hepatic insulin resistance via complex interplay of several metabolic pathways, at least some of which are independent of increased weight gain and caloric intake. The current evidence shows that the fructose, but not glucose, component of dietary sugar drives metabolic complications and contradicts the notion that fructose is merely a source of palatable calories that leads to increased weight gain and insulin resistance.