Matthew R. Jackman

Opposing effects of fructokinase C and A isoforms on fructose-induced metabolic syndrome in mice

Fructose intake from added sugars correlates with the epidemic rise in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. Fructose intake also causes features of metabolic syndrome in laboratory animals and humans. The first enzyme in fructose metabolism is fructokinase, which exists as two isoforms, A and C. Here we show that fructose-induced metabolic syndrome is prevented in mice lacking both isoforms but is exacerbated in mice lacking fructokinase A. Fructokinase C is expressed primarily in liver, intestine, and kidney and has high affinity for fructose, resulting in rapid metabolism and marked ATP depletion. In contrast, fructokinase A is widely distributed, has low affinity for fructose, and has less dramatic effects on ATP levels. By reducing the amount of fructose for metabolism in the liver, fructokinase A protects against fructokinase C-mediated metabolic syndrome. These studies provide insights into the mechanisms by which fructose causes obesity and metabolic syndrome.

High‐fat and high‐sucrose (western) diet induces steatohepatitis that is dependent on fructokinase

Fructose intake from added sugars has been implicated as a cause of nonalcoholic fatty liver disease. Here we tested the hypothesis that fructose may interact with a high‐fat diet to induce fatty liver, and to determine if this was dependent on a key enzyme in fructose metabolism, fructokinase. Wild‐type or fructokinase knockout mice were fed a low‐fat (11%), high‐fat (36%), or high‐fat (36%) and high‐sucrose (30%) diet for 15 weeks. Both wild‐type and fructokinase knockout mice developed obesity with mild hepatic steatosis and no evidence of hepatic inflammation on a high‐fat diet compared to a low‐fat diet. In contrast, wild‐type mice fed a high‐fat and high‐sucrose diet developed more severe hepatic steatosis with low‐grade inflammation and fibrosis, as noted by increased CD68, tumor necrosis factor alpha, monocyte chemoattractant protein‐1, alpha‐smooth muscle actin, and collagen I and TIMP1 expression. These changes were prevented in the fructokinase knockout mice. Conclusion: An additive effect of high‐fat and high‐sucrose diet on the development of hepatic steatosis exists. Further, the combination of sucrose with high‐fat diet may induce steatohepatitis. The protection in fructokinase knockout mice suggests a key role for fructose (from sucrose) in this development of steatohepatitis. These studies emphasize the important role of fructose in the development of fatty liver and nonalcoholic steatohepatitis. (Hepatology 2013;58:1632–1643)

Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo.

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential. Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1. These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo.

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.

Weight regain after sustained weight reduction is accompanied by suppressed oxidation of dietary fat and adipocyte hyperplasia

A dual-tracer approach (dietary 14C-palmitate and intraperitoneal 3H-H2O) was used to assess the trafficking of dietary fat and net retention of carbon in triglyceride depots during the first 24 h of weight regain. Obesity-prone male Wistar rats were allowed to mature under obesogenic conditions for 16 wk. One group was switched to ad libitum feeding of a low-fat diet for 10 wk (Obese group). The remaining rats were switched to an energy-restricted, low-fat diet for 10 wk that reduced body weight by 14% and were then assessed in energy balance (Reduced group), with free access to the low-fat diet (Relapse-Day1 group), or with a provision that induced a minor imbalance (+10 kcal) equivalent to that observed in obese rats (Gap-Matched group). Fat oxidation remained at a high, steady rate throughout the day in Obese rats, but was suppressed in Reduced, Gap-Matched, and Relapse-Day1 rats though 9, 18, and 24 h, respectively. The same caloric excess in Obese and Gap-Matched rats led to less fat oxidation over the day and greater trafficking of dietary fat to visceral depots in the latter. In addition to trafficking nutrients to storage, Relapse-Day1 rats had more small, presumably new, adipocytes at the end of 24 h. Dietary fat oxidation at 24 h was related to the phosphorylation of skeletal muscle acetyl-CoA carboxylase and fatty acid availability. These observations provide evidence of adaptations in the oxidation and trafficking of dietary fat that extend beyond the energy imbalance, which facilitate rapid, efficient regain during the relapse to obesity.

Regular exercise attenuates the metabolic drive to regain weight after long-term weight loss

Weight loss is accompanied by several metabolic adaptations that work together to promote rapid, efficient regain. We employed a rodent model of regain to examine the effects of a regular bout of treadmill exercise on these adaptations. Obesity was induced in obesity-prone rats with 16 wk of high-fat feeding and limited physical activity. Obese rats were then weight reduced (approximately 14% of body wt) with a calorie-restricted, low-fat diet and maintained at that reduced weight for 8 wk by providing limited provisions of the diet with (EX) or without (SED) a daily bout of treadmill exercise (15 m/min, 30 min/day, 6 days/wk). Weight regain, energy balance, fuel utilization, adipocyte cellularity, and humoral signals of adiposity were monitored during eight subsequent weeks of ad libitum feeding while the rats maintained their respective regimens of physical activity. Regular exercise decreased the rate of regain early in relapse and lowered the defended body weight. During weight maintenance, regular exercise reduced the biological drive to eat so that it came closer to matching the suppressed level of energy expenditure. The diurnal extremes in fuel preference observed in weight-reduced rats were blunted, since exercise promoted the oxidation of fat during periods of feeding (dark cycle) and promoted the oxidation of carbohydrate (CHO) later in the day during periods of deprivation (light cycle) . At the end of relapse, exercise reestablished the homeostatic steady state between intake and expenditure to defend a lower body weight. Compared with SED rats, relapsed EX rats exhibited a reduced turnover of energy, a lower 24-h oxidation of CHO, fewer adipocytes in abdominal fat pads, and peripheral signals that overestimated their adiposity. These observations indicate that regimented exercise altered several metabolic adaptations to weight reduction in a manner that would coordinately attenuate the propensity to regain lost weight.

The role for adipose tissue in weight regain after weight loss

Weight regain after weight loss is a substantial challenge in obesity therapeutics. Dieting leads to significant adaptations in the homeostatic system that controls body weight, which promotes overeating and the relapse to obesity. In this review, we focus specifically on the adaptations in white adipose tissues that contribute to the biological drive to regain weight after weight loss. Weight loss leads to a reduction in size of adipocytes and this decline in size alters their metabolic and inflammatory characteristics in a manner that facilitates the clearance and storage of ingested energy. We present the hypothesis whereby the long‐term signals reflecting stored energy and short‐term signals reflecting nutrient availability are derived from the cellularity characteristics of adipose tissues. These signals are received and integrated in the hypothalamus and hindbrain and an energy gap between appetite and metabolic requirements emerges and promotes a positive energy imbalance and weight regain. In this paradigm, the cellularity and metabolic characteristics of adipose tissues after energy‐restricted weight loss could explain the persistence of a biological drive to regain weight during both weight maintenance and the dynamic period of weight regain.

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.

Energy expenditure in obesity-prone and obesity-resistant rats before and after the introduction of a high-fat diet

While most rats gain weight when placed on a high-fat diet (HFD), some strains resist HFD-induced weight gain. To maintain weight, obesity-resistant (OR) rats must either eat less than obesity-prone (OP) rats or increase total energy expenditure (TEE). To determine if changes in TEE predispose to or protect from weight gain, energy expenditure, energy intake, and weight gain were measured in male and female OP and OR rats consuming a low-fat diet (LFD) and for 5 days after switching to a HFD. After 5 days on a HFD, OP rats gained significantly more weight (male: 42.8 ± 6.9 g, female: 25.5 ± 3.0 g) than their OR counterparts (male: 24.0 ± 7.5 g, female: 13.7 ± 1.4 g). Both male and female rats significantly increased their energy intake when transitioned to the HFD, and TEE increased modestly in all groups. Compared with female OP rats, female OR rats had a significantly greater increase in TEE on the HFD. This was due to an increase in both resting and nonresting energy expenditure. In contrast, the effect of the HFD in males was minor. TEE was also measured in female rats consuming a HFD, pair fed to LFD calories. The increase in TEE of pair-fed female OR rats was substantially less than what was seen in the HFD ad libitum condition. Physical activity was also measured in female rats. There was no evidence that increases in physical activity were the cause of the increased TEE seen in female OR rats consuming a HFD. These results suggest that resistance to HFD-induced weight gain in female OR rats may be due in part to an increase in TEE and a greater reliance on lipid as an energy source. Changes in TEE appear to be triggered by overconsumption of the HFD and not simply the diet composition.

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.