Ravindra Dhir

Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration

Fatty liver disease is associated with obesity and type 2 diabetes, and hepatic lipid accumulation may contribute to insulin resistance. Histone deacetylase 3 (Hdac3) controls the circadian rhythm of hepatic lipogenesis. Here we show that, despite severe hepatosteatosis, mice with liver-specific depletion of Hdac3 have higher insulin sensitivity without any changes in insulin signaling or body weight compared to wild-type mice. Hdac3 depletion reroutes metabolic precursors towards lipid synthesis and storage within lipid droplets and away from hepatic glucose production. Perilipin 2, which coats lipid droplets, is markedly induced upon Hdac3 depletion and contributes to the development of both steatosis and improved tolerance to glucose. These findings suggest that the sequestration of hepatic lipids in perilipin 2–coated droplets ameliorates insulin resistance and establish Hdac3 as a pivotal epigenomic modifier that integrates signals from the circadian clock in the regulation of hepatic intermediary metabolism.

The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity

Background:Myostatin, also known as Growth and Differentiation Factor 8, is a secreted protein that inhibits muscle growth. Disruption of myostatin signaling increases muscle mass and decreases glucose, but it is unclear whether these changes are related. We treated mice on chow and high-fat diets with a soluble activin receptor type IIB (ActRIIB, RAP-031), which is a putative endogenous signaling receptor for myostatin and other ligands of the TGF-β superfamily.Results:After 4 weeks, RAP-031 increased lean and muscle mass, grip strength and contractile force. RAP-031 enhanced the ability of insulin to suppress glucose production under clamp conditions in high-fat fed mice, but did not significantly change insulin-mediated glucose disposal. The hepatic insulin-sensitizing effect of RAP-031 treatment was associated with increased adiponectin levels. RAP-031 treatment for 10 weeks further increased muscle mass and drastically reduced fat content in mice on either chow or high-fat diet. RAP-031 suppressed hepatic glucose production and increased peripheral glucose uptake in chow-fed mice. In contrast, RAP-031 suppressed glucose production with no apparent change in glucose disposal in high-fat-diet mice.Conclusion:Our findings show that disruption of ActRIIB signaling is a viable pharmacological approach for treating obesity and diabetes.

Inhibition of ADRP prevents diet-induced insulin resistance

Diets with high fat content induce steatosis, insulin resistance, and type 2 diabetes. The lipid droplet protein adipose differentiation-related protein (ADRP) mediates hepatic steatosis, but whether this affects insulin action in the liver or peripheral organs in diet-induced obesity is uncertain. We fed C57BL/6J mice a high-fat diet and simultaneously treated them with an antisense oligonucleotide (ASO) against ADRP for 4 wk. Glucose homeostasis was assessed with clamp and tracer techniques. ADRP ASO decreased the levels of triglycerides and diacylglycerol in the liver, but fatty acids, long-chain fatty acyl CoAs, ceramides, and cholesterol were unchanged. Insulin action in the liver was enhanced after ADRP ASO treatment, whereas muscle and adipose tissue were not affected. ADRP ASO increased the phosphorylation of insulin receptor substrate (IRS)1, IRS2, and Akt, and decreased gluconeogenic enzymes and PKCepsilon, consistent with its insulin-sensitizing action. These results demonstrate an important role for ADRP in the pathogenesis of diet-induced insulin resistance.

CRTC2 (TORC2) Contributes to the Transcriptional Response to Fasting in the Liver but Is Not Required for the Maintenance of Glucose Homeostasis

The liver contributes to glucose homeostasis by promoting either storage or production of glucose, depending on the physiological state. The cAMP response element-binding protein (CREB) is a principal regulator of genes involved in coordinating the hepatic response to fasting, but its mechanism of gene activation remains controversial. We derived CRTC2 (CREB-regulated transcription coactivator 2, previously TORC2)-deficient mice to assess the contribution of this cofactor to hepatic glucose metabolism in vivo. CRTC2 mutant hepatocytes showed reduced glucose production in response to glucagon, which correlated with decreased CREB binding to several gluconeogenic genes. However, despite attenuated expression of CREB target genes, including PEPCK, G6Pase, and PGC-1alpha, no hypoglycemia was observed in mutant mice. Collectively, these results provide genetic evidence supporting a role for CRTC2 in the transcriptional response to fasting, but indicate only a limited contribution of this cofactor to the maintenance of glucose homeostasis.

Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice

Lipid droplets in the liver are coated with the perilipin family of proteins, notably adipocyte differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47). ADRP is increased in hepatic steatosis and is associated with hyperlipidemia, insulin resistance, and glucose intolerance. We have shown that reducing ADRP in the liver via antisense oligonucleotide (ASO) treatment attenuates steatosis and improves insulin sensitivity and glucose tolerance. We hypothesized that TIP47 has similar effects on hepatic lipid and glucose metabolism. We found that TIP47 mRNA and protein levels were increased in response to a high-fat diet (HFD) in C57BL/6J mice. TIP47 ASO treatment decreased liver TIP47 mRNA and protein levels without altering ADRP levels. Low-dose TIP47 ASO (15 mg/kg) and high-dose TIP47 ASO (50 mg/kg) decreased triglyceride content in the liver by 35% and 52%, respectively. Liver histology showed a drastic reduction in hepatic steatosis following TIP47 ASO treatment. The high dose of TIP47 ASO significantly blunted hepatic triglyceride secretion, improved glucose tolerance, and increased insulin sensitivity in liver, adipose tissue, and muscle. These findings show that TIP47 affects hepatic lipid and glucose metabolism and may be a target for the treatment of nonalcoholic fatty liver and related metabolic disorders.

Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats

Humans as well as other mammals experience an aging-related decline in drug metabolism as well as a diminution in growth hormone secretion. In the case of rats, these events are more pronounced in senescent males, whose expression of male-specific isoforms of cytochrome P450, the major drug-metabolizing enzymes and constituting ≈60-70% of the total cytochrome P450 in male rat liver, is completely suppressed, whereas female-dependent isoforms are remarkably induced to female-like levels. Overlooked in these independently reported studies is the fact that “signals” inherent in the masculine episodic and female continuous growth hormone profiles regulate expression and/or suppression of the dozen or so sex-dependent cytochrome P450 isoforms in rat liver. Whereas previous studies identified profound reductions in the pulse amplitudes of the masculine growth hormone profile as the cause for the diminished hormone secretion during aging, pulse heights are not recognized by the cytochromes as regulatory signals. Instead, we have shown that just a nominal secretion of growth hormone during the usual growth hormone-devoid interpulse period in the masculine episodic profile can explain the complete repression of male-specific CYP2C11, CYP3A2, and CYP2A2 and induction of female-dependent CYP2C12, CYP2C6, and CYP2A1 observed in senescent male rats.

Effects of perilipin 2 antisense oligonucleotide treatment on hepatic lipid metabolism and gene expression

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. We previously showed that Perilipin 2 (Plin2), a member of lipid droplet protein family, is markedly increased in fatty liver, and its reduction in the liver of diet-induced obese mice by antisense oligonucleotide (ASO) decreased steatosis and enhanced insulin sensitivity. Plin2-ASO treatment markedly suppressed lipogenic gene expression. To gain a better understanding of the biological role of Plin2 in liver, we performed microarray analysis to determine genes differentially regulated by Plin2-ASO compared with a control (scrambled) oligonucleotide (Cont). Male C57BL/6J mice on a high-fat diet were treated with Plin2- or Cont-ASO for 4 wk. Plin2-ASO decreased hepatic triglycerides, and this was associated with changes in expression of 1,363 genes. We analyzed the data for functional clustering and validated the expression of representative genes using real-time PCR. On the high-fat diet, Plin2-ASO decreased the expression of enzymes involved in fatty acid metabolism (acsl1, lipe) and steroid metabolism (hmgcr, hsd3b5, hsd17b2), suggesting that Plin2 affects hepatic lipid metabolism at the transcriptional level. Plin2-ASO also increased the expression of genes involved in regulation of hepatocyte proliferation (afp, H19), mitosis (ccna2, incenp, sgol1), and extracellular matrix (col1a1, col3a1, mmp8). Plin2-ASO had similar effects on gene expression in chow-fed mice. Together, these results indicate that Plin2 has diverse metabolic and structural roles in the liver, and its downregulation promotes hepatic fibrosis and proliferation.

Metabolic dysfunction associated with adiponectin deficiency enhances kainic acid-induced seizure severity

Metabolic syndrome has deleterious effects on the CNS, and recent evidence suggests that obesity rates are higher at presentation in children who develop epilepsy. Adiponectin is secreted by adipose tissue and acts in the brain and peripheral organs to regulate glucose and lipid metabolism. Adiponectin deficiency predisposes toward metabolic syndrome, characterized by obesity, insulin resistance, impaired glucose tolerance, hyperlipidemia, and cardiovascular morbidity. To investigate the relationship between metabolic syndrome and seizures, wild-type C57BL/6J and adiponectin knock-out mice were fed a high-fat diet, followed by treatment with low doses of kainic acid to induce seizures. Adiponectin deficiency in mice fed a high-fat diet resulted in greater fat accumulation, impaired glucose tolerance, hyperlipidemia, increased seizure severity, and increased hippocampal pathology. In contrast, there were no adverse effects of adiponectin deficiency on metabolic phenotype or seizure activity in mice fed a normal (low-fat) chow diet. These findings demonstrate that metabolic syndrome modulates the outcome of seizures and brain injury.

Estrogen sulfotransferase regulates body fat and glucose homeostasis in female mice

Estrogen regulates fat mass and distribution and glucose metabolism. We have previously found that estrogen sulfotransferase (EST), which inactivates estrogen through sulfoconjugation, was highly expressed in adipose tissue of male mice and induced by testosterone in female mice. To determine whether inhibition of estrogen in female adipose tissue affects adipose mass and metabolism, we generated transgenic mice expressing EST via the aP2 promoter. As expected, EST expression was increased in adipose tissue as well as macrophages. Parametrial and subcutaneous inguinal adipose mass and adipocyte size were significantly reduced in EST transgenic mice, but there was no change in retroperitoneal or brown adipose tissue. EST overexpression decreased the differentiation of primary adipocytes, and this was associated with reductions in the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, hormone-sensitive lipase, lipoprotein lipase, and leptin. Serum leptin levels were significantly lower in EST transgenic mice, whereas total and high-molecular-weight adiponectin levels were not different in transgenic and wild-type mice. Glucose uptake was blunted in parametrial adipose tissue during hyperinsulinemic-euglycemic clamp in EST transgenic mice. In contrast, hepatic insulin sensitivity was improved but muscle insulin sensitivity did not change in EST transgenic mice. These results reveal novel effects of EST on adipose tissue and glucose homeostasis in female mice.

Attenuated Expression of Episodic Growth Hormone-Induced CYP2C11 in Female Rats Associated with Suboptimal Activation of the Jak2/Stat5B and Other Modulating Signaling Pathways

Inherent sex differences in various parameters of growth, musculoskeletal function, metabolism, and cytochrome P450 (P450)-dependent drug metabolism have been reported in rats and humans administered typical intermittent/episodic growth hormone (GH) replacement therapy. Having infused and monitored the identical physiologic masculine (episodic) growth hormone profile to both hypophysectomized male and female rats, we observed that induction levels of hepatic CYP2C11 were 35 to 40% lower in females. Associated with the reduced expression of the P450 isoform in the episodic GH-treated females were dramatically lower activation levels of Janus kinase (Jak2), signal transducers and activators of transcription (Stat5A and 5B) as well as 50% less binding of Stat5B to the CYP2C11 promoter. Because the Jak2/Stat5B signaling pathway mediates the effects of the masculine GH profile on its target cells, we conclude that the lower induction level of CYP2C11 in females exposed to the masculine GH profile is probably due, at least in part, to the suboptimum activation of the Jak2/Stat5B pathway. In addition to the reduced activation of the Jak2/Stat5B pathway, we observed lower activational levels of mitogen-activated protein kinase (p44/p42) and, indirectly, nuclear factor-κB in the episodic GH-treated females that may be involved in attenuating the activity of the Jak2/Stat5B pathway diminishing CYP2C11 expression levels.