Elise S. Bales

Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease

The cytoplasmic lipid droplet (CLD) protein perilipin-2 (Plin2) is expressed in multiple nonadipose tissues, where it is thought to play a role in regulating their lipid storage properties. However, the extent to which Plin2 functions in nutrient utilization and metabolism, or how it influences the consequences of over-feeding, remains unclear. In this study, we demonstrate that the absence of Plin2 prevents high-fat diet(HFD)-induced obesity in male and female mice. This response is associated with increased formation of subcutaneous beige adipocyte cells with uncoupling protein 1 expression, and amelioration of inflammatory foci formation in white adipose tissue and steatosis in the liver. Experiments demonstrate that Plin2 loss results in reduced energy intake and increased physical activity in response to HFD feeding. Our study provides the first evidence that Plin2 contributes to HFD-induced obesity by modulating food intake, and that its absence prevents obesity-associated adipose tissue inflammatory foci and liver steatosis.

Endogenous fructose production and metabolism in the liver contributes to the development of metabolic syndrome

Carbohydrates with high glycemic index are proposed to promote the development of obesity, insulin resistance and fatty liver, but the mechanism by which this occurs remains unknown. High serum glucose concentrations glucose are known to induce the polyol pathway and increase fructose generation in the liver. Here we show that this hepatic, endogenously-produced fructose causes systemic metabolic changes. We demonstrate that mice unable to metabolize fructose are protected from an increase in energy intake and body weight, visceral obesity, fatty liver, elevated insulin levels and hyperleptinemia after exposure to 10% glucose for 14 weeks. In normal mice, glucose consumption is accompanied by aldose reductase and polyol pathway activation in steatotic areas. In this regard, we show that aldose reductase deficient mice were protected against glucose-induced fatty liver. We conclude that endogenous fructose generation and metabolism in the liver represents an important mechanism whereby glucose promotes the development of metabolic syndrome.

Mammary glands of adipophilin-null mice produce an amino-terminally truncated form of adipophilin that mediates milk lipid droplet formation and secretion

Adipophilin (ADPH), a member of the perilipin family of lipid droplet-associated proteins, is hypothesized to mediate milk lipid formation and secretion. Unexpectedly, the fat content of milk from ADPH-null mice was only modestly lower than that of wild-type controls, and neither TIP47 nor perilipin appeared to fully compensate for ADPH loss. This prompted us to investigate the possibility that the mutated ADPH gene was not a genuine null mutation. ADPH transcripts were detected in ADPH-null mammary tissue by quantitative real-time PCR, and C-terminal-specific, but not N-terminal-specific, ADPH antibodies detected a single lower molecular weight product and immunostained cytoplasmic lipid droplets (CLDs) and secreted milk fat globules in ADPH-null mammary tissue. Furthermore, stable cell lines expressing cDNA constructs corresponding to the ADPH-null mutation produced a product comparable in size to the one detected in ADPH-null mammary glands and localized to CLDs. Based on these data, we conclude that ADPH-null mice express an N-terminally truncated form of ADPH that retains the ability to promote the formation and secretion of milk lipids.

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.

Impact of High-Fat Diet and Obesity on Energy Balance and Fuel Utilization During the Metabolic Challenge of Lactation

The effects of obesity and a high‐fat (HF) diet on whole body and tissue‐specific metabolism of lactating dams and their offspring were examined in C57/B6 mice. Female mice were fed low‐fat (LF) or HF diets before and throughout pregnancy and lactation. HF‐fed mice were segregated into lean (HF‐Ln) and obese (HF‐Ob) groups before pregnancy by their weight gain response. Compared to LF‐Ln dams, HF‐Ln, and HF‐Ob dams exhibited a greater positive energy balance (EB) and increased dietary fat retention in peripheral tissues (P < 0.05). HF‐Ob dams had greater dietary fat retention in liver and adipose compared to HF‐Ln dams (P < 0.05). De novo synthesized fat was decreased in tissues and milk from HF‐fed dams compared to LF‐Ln dams (P < 0.05). However, less dietary and de novo synthesized fat was found in the HF‐Ob mammary glands compared to HF‐Ln (P < 0.05). Obesity was associated with reduced milk triglycerides relative to lean controls (P < 0.05). Compared to HF diet alone obesity has additional adverse affects, impairing both lipid metabolism as well as milk fat production. Growth rates of LF‐Ln litters were lower than HF‐Ln and HF‐Ob litters (P < 0.05). Total energy expenditure (TEE) of HF‐Ob litters was reduced relative to HF‐Ln litters, whereas their respiratory exchange ratios (RERs) were increased (P < 0.05). Collectively these data show that consumption of a HF diet significantly affects maternal and neonatal metabolism and that maternal obesity can independently alter these responses.

Chronic ethanol consumption in mice alters hepatocyte lipid droplet properties.

BACKGROUND Hepatosteatosis is a common pathological feature of impaired hepatic metabolism following chronic alcohol consumption. Although often benign and reversible, it is widely believed that steatosis is a risk factor for development of advanced liver pathologies, including steatohepatitis and fibrosis. The hepatocyte alterations accompanying the initiation of steatosis are not yet clearly defined. METHODS Induction of hepatosteatosis by chronic ethanol consumption was investigated using the Lieber-DeCarli (LD) high fat diet model. Effects were assessed by immunohistochemistry and blood and tissue enzymatic assays. Cell culture models were employed for mechanistic studies. RESULTS Pair feeding mice ethanol (LD-Et) or isocaloric control (LD-Co) diets for 6 weeks progressively increased hepatocyte triglyceride accumulation in morphological, biochemical, and zonally distinct cytoplasmic lipid droplets (CLD). The LD-Et diet induced zone 2-specific triglyceride accumulation in large CLD coated with perilipin, adipophilin (ADPH), and TIP47. In LD-Co-fed mice, CLD were significantly smaller than those in LD-Et-fed mice and lacked perilipin. A direct role of perilipin in formation of large CLD was further suggested by cell culture studies showing that perilipin-coated CLD were significantly larger than those coated with ADPH or TIP47. LD-Co- and LD-Et-fed animals also differed in hepatic metabolic stress responses. In LD-Et but not LD-Co-fed mice, inductions were observed in the following: microsomal ethanol-oxidizing system [cytochrome P-4502E1 (CYP2E1)], hypoxia response pathway (hypoxia-inducible factor 1 alpha, HIF1α), endoplasmic reticulum stress pathway (calreticulin), and synthesis of lipid peroxidation products [4-hydroxynonenal (4-HNE)]. CYP2E1 and HIF1 α immunostaining localized to zone 3 and did not correlate with accumulation of large CLD. In contrast, calreticulin and 4-HNE immunostaining closely correlated with large CLD accumulation. Importantly, 4-HNE staining significantly colocalized with ADPH and perilipin on the CLD surface. CONCLUSIONS These data suggest that ethanol contributes to macrosteatosis by both altering CLD protein composition and inducing lipid peroxide adduction of CLD-associated proteins.

Multiple functions encoded by the N-terminal PAT domain of adipophilin.

Adipophilin (ADPH), a member of the perilipin family of cytoplasmic lipid droplet (CLD)-binding proteins, is crucially dependent on triglyceride synthesis for stability. We have used cell lines expressing full-length or N-terminally modified forms of ADPH to investigate the role of the N-terminus in regulating ADPH stability and interactions with CLD. Full-length ADPH was unstable and could not be detected on CLDs unless cultures were incubated with oleic acid (OA) to stimulate triglyceride synthesis, or were treated with MG132 to block proteasomal degradation. By contrast, ADPH lacking amino acids 1-89 (Δ 2,3 ADPH), or N-terminally GFP-tagged full-length ADPH, was stable in the absence of OA or MG132, as was the closely related protein TIP47. However, none of these proteins localized to CLDs unless OA was added to the culture medium. Furthermore, immunofluorescence analysis showed that TIP47 localization to CLDs was prevented by full-length ADPH, but not by Δ 2,3 ADPH. These results suggest that the N-terminal region of ADPH mediates proteasomal degradation and access of TIP47 to the CLD surface and possibly contributes to CLD stability. Chimeras of ADPH and TIP47, generated by swapping their N- and C-terminal halves, showed that these properties are specific to ADPH.

Perilipin-2 Modulates Lipid Absorption and Microbiome Responses in the Mouse Intestine

Obesity and its co-morbidities, such as fatty liver disease, are increasingly prevalent worldwide health problems. Intestinal microorganisms have emerged as critical factors linking diet to host physiology and metabolic function, particularly in the context of lipid homeostasis. We previously demonstrated that deletion of the cytoplasmic lipid drop (CLD) protein Perilipin-2 (Plin2) in mice largely abrogates long-term deleterious effects of a high fat (HF) diet. Here we test the hypotheses that Plin2 function impacts the earliest steps of HF diet-mediated pathogenesis as well as the dynamics of diet-associated changes in gut microbiome diversity and function. WT and perilipin-2 null mice raised on a standard chow diet were randomized to either low fat (LF) or HF diets. After four days, animals were assessed for changes in physiological (body weight, energy balance, and fecal triglyceride levels), histochemical (enterocyte CLD content), and fecal microbiome parameters. Plin2-null mice had significantly lower respiratory exchange ratios, diminished frequencies of enterocyte CLDs, and increased fecal triglyceride levels compared with WT mice. Microbiome analyses, employing both 16S rRNA profiling and metagenomic deep sequencing, indicated that dietary fat content and Plin2 genotype were significantly and independently associated with gut microbiome composition, diversity, and functional differences. These data demonstrate that Plin2 modulates rapid effects of diet on fecal lipid levels, enterocyte CLD contents, and fuel utilization properties of mice that correlate with structural and functional differences in their gut microbial communities. Collectively, the data provide evidence of Plin2 regulated intestinal lipid uptake, which contributes to rapid changes in the gut microbial communities implicated in diet-induced obesity.

Maternal obesity during lactation may protect offspring from high fat diet-induced metabolic dysfunction

Background/ObjectivesThe current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother’s dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring.Subjects/MethodsTo determine if changes in the mother’s diet and adiposity have long-term effects on the baby’s metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother’s adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function.ResultsIn this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet.ConclusionExposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.